MX2014006733A - Antibodies for epidermal growth factor receptor 3 (her3) directed to domain ii of her3. - Google Patents

Abstract

The present invention relates to antibodies or fragments thereof that target an epitope of a HER3 receptor residing in domain 2 of the HER3 receptor to block both ligand-dependent and ligand-independent signal transduction and tumor growth; and compositions and methods of use thereof.

Description

ANTIBODIES FOR THE FACTOR RECEIVER EPIDERMAL GROWTH 3 (HER3) DIRECTED TO DOMAIN II FROM HER3 Related Requests This application claims priority for the Provisional Patent Application of the United States of America Number 61 / 566,912 filed on December 5, 201 1, the content of which is hereby incorporated by reference in its entirety.

Field of the Invention This invention relates, in general terms, to antibodies or fragments thereof that recognize an HER3 epitope, which comprises residues within domain 2 which results in the inhibition of signal transduction both ligand-dependent and independent of the ligand, and tumor growth; and to compositions and methods of using these antibodies or fragments thereof.

Background of the Invention The human epidermal growth factor receptor 3 (ErbB3, also known as HER3) is a receptor tyrosine protein kinase and belongs to the subfamily of receptor tyrosine protein kinases of the epidermal growth factor receptor (EGFR), which also includes EGFR (HER1, ErbB1), HER2 (ErbB2, Neu), and HER4 (ErbB4) (Plowman et al. (1990) Proc. Nati. Acad. Sci. USA 87: 4905-4909; Kraus et al. (1989) Proc.

Nati Acad. Sci. USA 86: 9193-9197; and Kraus et al. (1993) Proc. Nati Acad. Sci. USA 90: 2900-2904). As the prototypic epidermal growth factor receptor, the transmembrane HER3 receptor consists of an extracellular ligand binding domain (ECD), a dimerization domain within the extracellular ligand binding domain (ECD), a transmembrane domain, a transmembrane domain. type intracellular tyrosine protein kinase (TKD), and a C-terminal phosphorylation domain. Unlike the other members of the HER family, the H 3 ER3 kinase domain exhibits very low intrinsic kinase activity.

Ligands of Neuregulin 1 (NRG) or Neuregulin 2 bind to the extracellular domain of HER3 and activate the pathway of signaling mediated by the receptor by promoting dimerization with other dimerization partners, such as HER2. The heterodimerization results in the activation and trans-phosphorylation of the intracellular domain of HER3, and is a means not only for the diversification of the signal, but also for the amplification of the signal. In addition, heterodimerization of HER3 can also occur in the absence of activating ligands, and this is commonly referred to as HER3 activation independent of the ligand. For example, when HER2 is expressed at high levels as a result of genetic amplification (for example, in breast, lung, ovarian or gastric cancer), spontaneous HER2 / HER3 dimers can be formed. In this situation, the HER2 / HER3 is considered as the signaling dimer of ErbB more active and, therefore, is highly transformative.

An increase of HER3 has been found in several types of cancer, such as breast, lung, gastromestinal and pancreatic cancers. Interestingly, a correlation between HER2 / H ER3 expression and progress from a non-invasive stage to an invasive stage has been demonstrated (Alimandi et al. (1995) Oncogene 10: 1813-1821; DeFazio et al. (2000) Cancer 87 : 487-498; Naidu et al. (1988) Br. J. Cancer 78: 1385-1390). In accordance with the above, agents that interfere with HER3-mediated signaling are needed.

Brief Description of the Invention The invention is based on the discovery of antibodies or fragments thereof which bind to an epitope (linear, non-linear, conformational) of the HER3 receptor comprising the amino acid residues within domain 2 of HER3. Surprisingly, the binding of antibodies or fragments thereof to an epitope within domain 2 of HER3 blocks the HER3 signaling pathways both ligand-dependent (eg, neuregulin) and ligand-independent.

In accordance with the foregoing, in one aspect, the invention pertains to an isolated antibody or fragment thereof that recognizes an epitope of a HER3 receptor, wherein the epitope comprises amino acid residues 208-328 within domain 2 of the HER3 receptor. , wherein the antibody or a fragment thereof recognizes at least the amino acid residue 268 within of domain 2, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent.

The epitope is selected from the group consisting of a linear epitope, a non-linear epitope, and a conformational epitope. In one embodiment, the antibody or a fragment thereof binds to an inactive state of the HER3 receptor. In one embodiment, the ligand of H ER3 that binds to the ligand binding site fails to activate the signal transduction of HER3. In one embodiment, a HER3 ligand can be linked in a concurrent manner to the ligand binding site on the H ER3 receptor. In one embodiment, the HER3 ligand is selected from the group consisting of neurregulin 1 (NRG), neurregulin 2, beta-cellulin, epidermal growth factor that binds to heparin, and epiregulin. The antibodies or fragments thereof described herein can be linked to amino acid residue 268 (within domain 2). In one embodiment, the linker amino acid 268 affects the bond in domain 2, thereby blocking the binding to the antibody or antibody fragment. In one embodiment, the antibody or a fragment thereof has a characteristic selected from the group consisting of destabilizing HER3 in such a way that it is susceptible to degradation, accelerating HER3 sub-regulation of the cell surface, inhibiting dimerization with other HER receptors, and generate an unnatural HER3 dimer that is susceptible to degradation proteolytic or that is unable to dimerize with other receptor tyrosine kinases. In one embodiment, binding of the antibody or a fragment thereof to the H ER3 receptor in the absence of a HER3 ligand reduces the ligand-independent formation of a H ER2-HER3 protein complex in a cell that expresses HER2 and HER3. In one embodiment, the H ER3 receptor fails to dimerize with the HER2 receptor to form a protein complex of HER2-HER3. In one embodiment, the failure to form a HER2-HER3 protein complex prevents the activation of signal transduction. In one embodiment, the antibody or a fragment thereof inhibits the phosphorylation of HER3 as assessed by an independent phosphorylation assay of the HER3 ligand. In one embodiment, the HER3 ligand-independent phosphorylation assay utilizes cells amplified by HER2, wherein cells amplified by HER2 are SK-Br-3 and BT-474 cells. In one embodiment, binding the antibody or a fragment thereof to the HER3 receptor in the presence of a HER3 ligand reduces the formation of a HER2-HER3 protein complex dependent on the ligand in a cell expressing HER2 and HER3. In one embodiment, the H ER3 receptor fails to dimerize with the HER2 receptor in the presence of a HER3 ligand to form a protein complex of HER2-HER3. In one embodiment, failure to form a protein complex of HER2-H ER3 prevents the activation of signal transduction. In one embodiment, the antibody or a fragment thereof inhibits the phosphorylation of HER3 as evaluated by the ligand-dependent phosphorylation assay of H ER3. In one embodiment, the HER3 ligand-dependent phosphorylation assay utilizes MCF7 cells stimulated in the presence of neurregulin (NRG). In one embodiment, the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, and a synthetic antibody.

In another aspect, the invention pertains to an isolated antibody or fragment thereof that recognizes an epitope of a HER3 receptor within domain 2 of the H receptor ER3, wherein the epitope comprises amino acid residues 208-328 within domain 2 of the HER3 receptor, wherein the antibody or a fragment thereof recognizes at least amino acid residue 268 within domain 2, and wherein the antibody or a fragment thereof has a dissociation (KD) of at least 1 x 107 M \ 108 M 1, 109 M 1, 101 ° M 1, 101 1 M 1, 1012 M 1, 1013 M 1, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent. In one embodiment, the antibody or a fragment thereof inhibits the phosphorylation of HER3 as measured by an in vitro phosphorylation assay selected from the group consisting of phospho-HER3 and phospho-Akt. In one embodiment, the antibody or a fragment thereof binds to the same epitope as an antibody described in Table 1. In one embodiment, the isolated antibody or the fragment thereof competes cross-wise with an antibody described in Table 1. In one embodiment, the fragment of an antibody is selected from the group consisting of: Fab, F (ab2)? F (ab) 2 ', scFv, VHH, VH, VL, dAbs.

In another aspect, the invention pertains to a pharmaceutical composition, which comprises an antibody or a fragment thereof and a pharmaceutically acceptable carrier, wherein the antibody or a fragment thereof binds to a HER3 receptor comprising amino acid residues 208 -328 within domain 2 of the HER3 receptor, wherein the antibody or fragment thereof recognizes at least amino acid residue 268 within domain 2, and wherein the antibody or a fragment thereof blocks signal transduction both dependent on the ligand as independent of the ligand. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent. In one embodiment, the additional therapeutic agent is selected from the group consisting of a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor and a PI3 kinase inhibitor. In one embodiment, the additional therapeutic agent is an HER1 inhibitor selected from the group consisting of Matuzumab (EMD72000), Erbitux® / Cetuximab, Vectibix® / Panitumumab, mAb 806, Nimotuzumab, Iressa® / Gefitinib, CI-1033 ( PD183805), Lapatinib (GW-572016), Tykerb® / Lapatinib Ditosylate, Tarceva® / Erlotinib HCL (OSI-774), PKI-166, and Tovok®; a HER2 inhibitor selected from the group consisting of Pertuzumab, Trastuzumab, MM-1 1 1, nepatinib, lapatinib or lapatinib ditosylate / Tykerb®; a HER3 inhibitor selected from the group consisting of MM-121, M M-1 1 1, IB4C3, 2DID12 (U3 Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEH D7945A (Genentech), MOR10703 (Novartis), and small molecules that inhibit HER3; and an H ER4 inhibitor. In one embodiment, the additional therapeutic agent is an mTOR inhibitor selected from the group consisting of Temsirolimus / Torisel®, ridaforolimus / Deforolimus, AP23573, MK8669, everolimus / Affinitor®. In one embodiment, the additional therapeutic agent is a PI3 kinase inhibitor selected from the group consisting of GDC 0941, BEZ235, BMK120 and BYL719.

In another aspect, the invention pertains to a method for the treatment of a cancer, which comprises selecting a subject having a cancer expressing HER3, administering to the subject, an effective amount of a composition comprising an antibody or a fragment thereof. as disclosed in Table 1, wherein the antibody or a fragment thereof recognizes an epitope of a HER3 receptor comprising amino acid residues 208-328 within domain 2 of the H receptor ER3, wherein the antibody or a fragment thereof recognizes at least the amino acid residue 268 within domain 2, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent. In one modality, the subject is a human being and the cancer is selects from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, carcinoma scaly cells, peripheral tumors of the nerve sheath, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophageal cancer, glioblastoma, clear soft tissue sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prosthetic hyperplasia (BPH), gynecomastia, and endometriosis. In one modality, cancer is breast cancer.

In one aspect, the invention pertains to an antibody or fragment thereof, for use in the treatment of a cancer mediated by a signal-dependent signal transduction or signal transduction pathway independent of the HER3 ligand. In one aspect, the invention pertains to an antibody or fragment thereof, for use as a medicament. In one aspect, the invention pertains to the use of an antibody or a fragment thereof for the manufacture of a medicament for the treatment of a cancer mediated by a ligand-dependent signal transduction pathway or signal transduction independent of the ligand of HER3 selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophageal cancer, glioblastoma, clear soft tissue sarcoma, malignant mesothelioma, neuro-fibromatosis, renal cancer, melanoma, prostate cancer, benign prosthetic hyperplasia (BPH), gynecomastia, and endometriosis. Brief Description of the Figures Figure 1: SET curves of representative MOR12616 and MOR12925 obtained with human HER3.

Figure 2: Determination of binding of SK-Br-3 cells by FACS titration.

Figure 3: HER3 domain binding ELISA titration curves.

Figure 4: Linker ELISA curves of the HER3 mutant.

Figure 5: Competition with the epitope of HER3 using ELISA Figure 6: Inhibition of HER3 and Akt phosphorylation induced by the ligand.

Figure 7: Inhibition of phosphorylation of HER3 and Akt independent of the ligand in cell lines amplified by HER2.

Figure 8: Inhibition of ligand-dependent cell proliferation (A) and ligand-independent (B, C).

Figure 9: Data showing the in vivo inhibition of tumor growth in BxPC3 (A) and BT474 (B).

Detailed description of the invention Definitions In order that the present invention can be more easily understood, certain terms are defined first. Throughout the detailed description, additional definitions are stipulated.

The phrase "signal transduction" or "signaling activity", as used herein, refers to a causal biochemical relationship initiated in general terms by the protein-protein interaction, such as the binding of a growth factor to a receptor, which results in the transmission of a signal from one portion of a cell to another portion of a cell.For HER3, transmission involves the specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in the series of reactions that cause signal transduction The penultimate processes typically include nuclear events, which result in a change in gene expression.

The term "HER3" or "HER3 receptor", also known as "ErbB3", as used herein, refers to a mammalian HER3 protein, and "her3" or "erbB3" refers to a her3 gene of mammal. The preferred HER3 protein is the human HER3 protein present in the cell membrane of a cell. The human her3 gene is described in the United States Patent of North America Number 5,480,968, and in Plowman et al. (1990) Proc. Nati Acad. Sci. USA, 87: 4905-4909.

Human HER3, as defined in Accession No. NP_001973 (human), is represented below as SEQ ID NO: 1. The entire nomenclature is for the full-length immature HER3 (amino acids 1 to 1342). Immature HER3 dissociate between 19 and 20 positions, which results in the protein of mature HER3 (from 20-1342 amino acids) mrandalqvl gllfslargs evgnsqavcp gtlnglsvtg daenqyqtly klyercevvm gnleivltgh nadlsflqwi revtgyvlva mnefstlplp nlrvvrgtqv ydgkfaifvm Inyntnssha Irqlrltqlt eilsggvyie kndklchmdt idwrdivrdr daeiwkdng rscppchevc kgrcwgpgse dcqtltktic apqenghcfg pnpnqcchde caggcsgpqd tdcfacrhfn dsgacvprcp qplvynkltf qlepnphtky qyggvcvasc phnfvvdqts cvracppdkm evdknglkmc epcgglcpka cegtgsgsrf qtvdssnidg fvnctkilgn Idflitglng dpwhkipald peklnvfrtv reitgylniq swpphmhnfs vfsnlttigg rslynrgfsl limknlnvts Igfrslkeis agriyisanr qlcyhhslnw tkvlrgptee rldikhnrpr rdcvaegkvc dplcssggcw gpgpgqclsc rnysrggvcv thcnflngep refaheaecf schpecqpme gtatcngsgs dtcaqcahfr dgphcvsscp hgvlgakgpi ykypdvqnec rpchenctqg ckgpelqdcl gqtlvligkt hltmaltvia glvvifmmlg gtflywrgrr iqnkramrry lergesiepl dpsekankvl arifketelr klkvlgsgvf gtvhkgvwip egesikipvc ikviedksgr qsfqavtdhm laigsldhah ivrllglcpg ss lqlvtqilo plgslldhvr qhrgalgpql llnwgvqiak gmyyleehgm vhrnlaarnv llkspsqvqv adfgvadllp pddkqllyse aktpikwmal esihfgkyth qsdvwsygvt vwelmtfgae pyaglrlaev pdllekgerl aqpqictidv ymvmvkcwmi denirptfke laneftrmar dpprylvikr esgpgiapgp ephgltnkkl eevelepeld Idldleaeed nlatttlgsa Islpvgtlnr prgsqsllsp ssgympmnqg nlgescqesa vsgssercpr pvslhpmprg clasessegh vtgseaelqe kvsmcrsrsr srsprprgds ayhsqrhsll tpvtplsppg leeedvngyv mpdthlkgtp ssregtlssv glssvlgtee ededecyeym nrrrrhspph pprpssleel gyeymdvgsd Isaslgstqs cplhpvpimp tagttpdedy eymnrqrdgg gpggdyaamg acpaseqgye emrafqgpgh qaphvhyarl ktlrsleatd safdnpdywh srlfpkanaq rt (SEQ ID NO: 1).

The "HER2-H ER3 protein complex" is a non-covalently associated oligomer that contains the HER2 receptor and the HER3 receptor. This complex can be formed when a cell expressing both of these receptors is exposed to a HER3 ligand, e.g., NRG, or when HER2 is active or over-expressed.

The phrase "HER3 activity" or "HER3 activation", as used herein, refers to an increase in oligomerization (e.g., an increase in complexes containing HER3), phosphorylation of HER3, conformational reconfigurations (for example, those induced by ligands), and in downstream signaling mediated by HER3.

The term "stabilization" or "stabilized", used in the context of HER3, refers to an antibody or fragment thereof that directly maintains (secures, binds, arrests, preferentially binds, favors) the inactive state or the conformation of HER3. without blocking the ligand binding to HER3, such that the ligand binding is no longer able to activate HER3.

The term "ligand-dependent signaling", as used herein, refers to the activation of HER3 by of the ligand. Activation of HER3 is evidenced by an increase in heterodimerization and / or phosphorylation of H ER3, such that the downstream signaling pathways are activated (eg, PI3K). The antibody or fragment thereof can reduce in a statistically significant manner the amount of phosphorylated HER3 in a stimulated cell exposed to an antibody or fragment thereof, relative to an untreated (control) cell, as measured using the assays. described in the Examples. The cell expressing HER3 can be a cell line that occurs naturally (eg, MCF7) or can be produced in a recombinant manner by introducing nucleic acids encoding the HER3 protein in a host cell. Cell stimulation can be presented either by the exogenous addition of an activating HER3 ligand, or by the endogenous expression of an activating ligand.

The antibody or fragment thereof that "reduces the activation of HER3 induced by neurregulin in a cell" is one that statistically reduces the tyrosine phosphorylation of HER3 relative to an untreated cell (control), as measured using the assays described in the Examples. This can be determined based on the phosphotyrosine levels of HER3 following the exposure of HER3 to the NRG and the antibody of interest. The cell expressing the HER3 protein can be a cell or cell line that occurs naturally (eg, MCF7) or can be produced in a recombinant.

The term "ligand-independent signaling", as used herein, refers to cellular HER3 activity (eg, phosphorylation), in the absence of a ligand binding requirement, eg, the activation of H ER3. ligand-independent can be a result of over-expression of HER2 or activating mutations in components of the HER3 heterodimer, such as EGFR and HER2.The antibody or fragment thereof can reduce the amount of HER3 in a statistically significant manner. phosphorylated in a cell exposed to an antibody or a fragment thereof, in relation to an untreated cell (control) The cell expressing HER3 may be a cell line that occurs naturally (eg, SK-Br-3) or it can be produced in a recombinant manner by the introduction of nucleic acids encoding the HER3 protein in a host cell.

The term "blocks", as used herein, refers to stopping or preventing an interaction or a process, for example, stopping ligand-dependent or ligand-independent signaling.

The term "recognize", as used herein, refers to an antibody or a fragment thereof that encounters and interacts (eg, binds) with its epitope in domain 2 of HER3, eg, an antibody or a fragment thereof that interacts with at least one amino acid residue within the HER3 domain 2 (amino acid residues 208-328 of SEQ ID NO: 1). In another example, the antibody or a fragment thereof that interacts with at least Lys 268 within domain 2 of HER3.

The phrase "binds in a concurrent manner," as used herein, refers to a ligand of HER3 that can bind to a ligand binding site on the HER3 receptor together with the HER3 antibody or a fragment of the This means that both the antibody and the ligand can bind to the HER3 receptor together For purposes of illustration only, the HER3 ligand N RG can bind to the HER3 receptor together with the H ER3 antibody. of the ligand and the antibody is described in the Examples section.

The term "fails", as used herein, refers to an antibody or fragment thereof that does not make a particular event, eg, an antibody or a fragment thereof that "fails to activate signal transduction". it is one that does not trigger signal transduction.

The term "antibody", as used herein, refers to an integer antibody that interacts with (e.g., binding, steric hindrance, stabilization / destabilization, spatial distribution) an epitope of HER3, and inhibits signal transduction. "antibody" that occurs naturally is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. comprised of a heavy chain variable region (abbreviated herein as VH), and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH 1, CH 2 and CH 3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL), and a light chain constant region. The light chain constant region is comprised of a domain, CL. The VH and VL regions can further be subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed structure regions (FR). Each VH and VL is composed of three complementarity determining regions (CDRs) and four structure regions (FRs) arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 . The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single chain Fvs (scFv), disulfide linked Fvs (sdFv), Fab fragments, fragments F (ab '), and anti-idiotypic antibodies (anti-ld) (including, example, anti-idiotypic antibodies (anti-ld) the antibodies of the invention), and fragments of binding to the epitope of any of the going . The antibodies can be of any type of isotype (e.g., I g G, I g E, IgM, IgD, IgA and I g Y), or isotype subclass (e.g., I g G 1, IgG2, IgG3, IgG4 , Ig A 1 and I g A2).

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both light chain (VL) and heavy chain (VH) portions determine the recognition and specificity of the antigen. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH 1, CH 2 or CH 3) confer important biological properties, such as secretion, transplacental mobility, binding to the Fe receptor, complement binding, and the like. By convention, the numbering of the domains of the constant regions increases as they become more distal from the antigen binding site or the amino terminus of the antibody. The N-terminus is a variable region, and the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxyl terminus of the heavy and light chain, respectively.

The phrase "antibody fragment," as used herein, refers to one or more portions of an antibody that retain the ability to interact specifically (eg, example, through linkage, steric impedance, stabilization / destabilization, spatial distribution) with an epitope of H ER3 and inhibit signal transduction. Examples of the fragments link a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; an F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the joint region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al. (1989) Nature 341: 544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).

Additionally, although the two domains of the Fv, VL and VH fragment are encoded by separate genes, they can be linked, using recombinant methods, by means of a synthetic linker that makes it possible to elaborate them as a single protein chain where the pair of VL regions and VH monovalent molecules (known as single chain Fv (scFv), see, example, Bird et al (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Nati. Acad. Sci. 85: 5879-5883). It is also intended that these single-chain antibodies be encompassed within the term "antibody fragment." These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened their usefulness of the same way that the antibodies intact.

Antibody fragments can also be incorporated into single domain antibodies, antibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, example, Hollinger and Hudson, (2005) Nature Biotechnology 23 : 1 126-1 136). Antibody fragments can be grafted onto scaffolds based on polypeptides, such as Fibronectin type II I (Fn3) (see U.S. Patent No. 6,703,199, which discloses monobodies of fibronectin polypeptides).

The antibody fragments can be incorporated into single chain molecules comprising a pair of Fv segments in a row (VH-CH 1 -VH-CH 1), which, together with the complementary light chain polypeptides, a pair of regions of antigen binding (Zapata et al. (1995) Protein Eng. 8: 1057-1062; and U.S. Patent Number 5,641, 870).

The term "epitope" includes any protein determinant capable of having a specific binding to an immunoglobulin or otherwise interacting with a molecule. Epitopic determinants in general terms consist of groups of chemically active surface molecules, such as amino acid or carbohydrate side chains or sugar chains, and may have specific three-dimensional structural characteristics, as well as specific loading characteristics. An epitope can be "linear", "non-linear", or "conformational." In one embodiment, the epitope is within domain 2 of HER 3. In one embodiment, the epitope is a linear epitope within domain 2 of HER 3. In one embodiment, the epitope is an epitope non-linear within domain 2 of HER 3. In another embodiment, the epitope is a conformational epitope comprising the amino acid residues within domain 2 of HER 3. In one embodiment, the epitope comprises at least one amino acid residue within domain 2. of HER3 (amino acids 208-328 of SEQ ID NO: 1), or a subset thereof In one embodiment, the epitope comprises at least the amino acid Lys268 (within domain 2) of SEQ ID NO: 1. antibodies or fragments thereof described herein may be linked to Lys268 within domain 2 of HER3.

The term "linear epitope" refers to an epitope with all points of interaction between the protein and the interacting molecule (such as an antibody) that occurs linearly along the primary amino acid sequence of the protein (i.e. continuous amino acids). Once a desired epitope on an antigen is determined, it is possible to generate antibodies for that epitope, for example, using the techniques described in the present invention. In an alternative way, during the discovery process, the generation and characterization of the antibodies can elucidate information about the desirable epitopes. From this information, then it is possible to track in a competitive way the antibodies to bind to it epitope. One approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind to each other, for example, antibodies compete for binding to the antigen. A high throughput process for "reserving" the antibodies based on their cross-competition is described in International Patent Application Number WO 2003/48731. As will be appreciated by one skilled in the art, virtually anything to which it can be linked specifically an antibody could be an epitope An epitope can comprise the residues to which the antibody binds.

The term "non-linear epitope" refers to an epitope with non-contiguous amino acids that form a three-dimensional structure within a particular domain (e.g., within domain 1, within domain 2, within domain 3, or within domain 4). ). In one embodiment, the non-linear epitope is within domain 2. The non-linear epitope can also occur between two or more domains (e.g., the interface between domains 3-4). The non-linear epitope also refers to the non-contiguous amino acids that are a result of a three-dimensional structure within a particular domain.

The term "conformational epitope" refers to an epitope wherein the discontinuous amino acids come together in a three-dimensional conformation that involves at least two different domains, such as domain 2 and domain 4; or domain 3 and domain 4. In a conformational epitope, the dots of interaction are presented through the amino acid residues that are separated from each other on the protein. As will be appreciated by a person skilled in the art, the space that is occupied by a residue or by a side chain that creates the shape of a molecule helps determine what an epitope is.

In general terms, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

The regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Volume 66 (Glenn E. Morris, Editor, 1996) Humana Press, Totowa, New Jerscy. For example, linear epitopes can be determined, for example, by concurrently synthesizing large numbers of peptides on solid supports, peptides corresponding to portions of the protein molecule, and reacting the peptides with the antibodies while the peptides are still attached to the supports. These techniques are known in the art, and are described, for example, in U.S. Patent No. 4,708,871; in Geysen et al. (1984) Proc. Nati Acad. Sci. USA 8: 3998-4002; in Geysen et al. (1985) Proc. Nati Acad. Sci. USA 82; 78-182; and in Geysen et al. (1986) Mol. Immunol. 23: 709-715.

In a similar manner, conformational epitopes are easily identified by determining the spatial conformation of amino acids, such as by, for example, hydrogen / deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra. The antigenic regions of the proteins can also be identified using conventional antigenicity and hydropathy plots, such as those calculated using, for example, the Omiga software program version 1.0 available from the Oxford Molecular Group. This computer program employs the method of Hopp / Woods, Hopp et al. (1981) Proc. Nati Acad. Sci USA 78: 3824-3828; to determine the antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al. (1982) J. Mol. Biol. 157: 105-132; for hydropathy charts.

The phrases "monoclonal antibody" or "monoclonal antibody composition", as used herein, refer to polypeptides, including antibodies, antibody fragments, bispecific antibodies, etc. having a substantially identical or derived amino acid sequence. from the same genetic source This term also includes the preparations of antibody molecules of a single molecular composition A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

The phrase "human antibody", as used herein, it includes antibodies that have variable regions in which both the structure regions (FR) and the complementarity determining regions (CDR) are derived from sequences of human origin. Additionally, if the antibody contains a constant region, the constant region is also derived from human sequences, for example, the human germline sequences, or the mutated versions of the human germline sequences, or the antibody contains consensus structure sequences derived from the analysis of human structure sequences, for example, as described in Knappik et al. (2000) J Mol Biol 296: 57-86). The structures and locations of the immunoglobulin variable domains, for example, complementarity determining regions (CDRs), can be defined using well-known numbering schemes, for example, the Kabat numbering scheme, the Chotia numbering scheme. , or a combination of Kabat and Chotia (see, for example, Sequences of Proteins of Immunological Interest, Ü.S. Department of Health and Human Services (1991), Kabat Publishers and collaborators; Lazikani et al. (1997) J. Mol. Bio. 273: 927-948); Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, N1H Publication No. 91 -3242 U.S. Department of Health and Human Services (Department of Health and Human Services of the United States); Chotia et al. (1987) J.

Mol. Biol. 196: 901-917; Chotia et al (1989) Nature 342: 877-883; and Al-Lazikani et al. (1997) J. Mol. Biol. 273: 927-948.

The human antibodies of the invention may include amino acid residues not encoded by human sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote processing stability ).

The phrase "human monoclonal antibody", as used herein, refers to antibodies that exhibit a single binding specificity, which have variable regions where both the structure regions (FR) and the complementarity determining regions ( CDR) are derived from human sequences In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B-cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, which has a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The phrase "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g. a mouse) that is transgenic or transchromosomal for the genes of human immunoglobulin or a hybridoma prepared therefrom, the antibodies isolated from a host cell transformed to express the human antibody, for example, from a transfectome, the antibodies isolated from a recombinant library of human antibodies of combination, and antibodies prepared, expressed, created or isolated by any other means that entails the splicing of all or a portion of a human immunoglobulin genetic sequence with other DNA sequences. Recombinant human antibodies have variable regions wherein the framework regions (FR) and the complementarity determining regions (CDRs) are derived from the human germline immunoglobulin sequences. In certain embodiments, however, recombinant human antibodies can be subjected to in vitro mutagenesis (or, when a transgenic animal is used for the human immunoglobulin (Ig) sequences, somatic mutagenesis in vivo) and, therefore, the sequences of amino acids of the VH and VL regions of the recombinant antibodies are the sequences that, although derived from, and are related to the VH sequences and a VL of the human germline, may not naturally exist within the antibody repertoire of the human germ line in vivo.

The specific link between two entities means a link with an equilibrium constant (KA) (kactivad / k desactivated) of at least 102M 1, at least 5x102M 1, at least 103M 1, at least 5x103M 1, at least 104M 1at least 5x104M \ at least 105M 1, at least 5x105M 1, at least 106M 1, at least 5x106M 1, at least 107M 1, at least 5x107M 1, at least 10®M 1, when less 5X108M 1, at least 109M \ at least 5x109M 1, at least 101 ° M 1, at least 5x101 ° M 1, at least 1011M 1, at least 5x1011M 1, at least 1012M 1, at least 5X1012M 1, at least 1013M 1, at least 5x1013 M 1, at least 1014M 1, at least 5x1014M 1, at least 1015M 1, or at least 5x101sM 1.

The phrase "binds specifically (or selectively)" refers to a binding reaction of an antibody that binds HER3 and the HER3 receptor in a heterogeneous population of proteins and other biological products. In addition to the equilibrium constant (KA) mentioned above, an antibody that binds to HER3 of the invention typically also has a dissociation index constant (KD) (kdeSactavad / kactivated) of less than 5x10 2 M, less than 102M, less than 5x103M, less than 103M, less than 5x104M, less than 104M, less than 5x10 sM, less than 105M, less than 5X106M, less than 106M, less than 5x107M, less than 107M, less than 5x10 sM , less than 108M, less than 5x109M, less than 109, less than 5x101 ° M, less than 101 ° M, less than 5x1011M, less than 1011M, less than 5x1012M, less than 10 to 12M, less than 5X10 13M, less than 1013M, less than 5x1014M, less than 1014M, less than 5x1015M, or less than 1015M or less, and bind to HER3 with an affinity that is at least two times greater than its affinity for binding to a non-specific antigen (e.g., human serum albumin (HSA)).

In one embodiment, the antibody or fragment thereof has a dissociation constant (Kd) of less than 3,000 pM, less than 2,500 pM, less than 2,000 pM, less than 1,500 pM, less than 1,000 pM, less 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 10 pM, or less than 1 pM, as evaluated using a method described herein or known to a person skilled in the art (for example, a BIAcore, ELISA, FACS, SET assay) (Biacore International AB, Uppsala, Sweden).

The term "Kasoc" or "Ka", as used herein, refers to the rate of association of a particular antibody-antigen interaction, while the term "Kdis" or "Kd", as used herein refers to the dissociation rate of a particular antibody-antigen interaction The term "KD", as used herein, refers to the dissociation constant, which is obtained from the ratio of Kd to Ka. (ie, Kd / Ka), and is expressed as a molar concentration (M.) KD values for antibodies can be determined using methods well established in the art.A method for determining the KD of an antibody is using resonance of surface plasmon, or using a biosensor system, such as a Biacore® system.

The term "affinity", as used herein, refers to to the force of interaction between the antibody and the antigen in the individual antigenic sites. Within each antigenic site, the variable region of the "arm" of the antibody interacts through weak non-covalent forces with antigen at numerous sites, the more interactions there are, the stronger the affinity.

The term "avidity", as used herein, refers to an informative measure of the overall stability or strength of the antibody-antigen complex.It is controlled by three major factors: antibody-epitope affinity, the valence of both the antigen Finally, these factors define the specificity of the antibody, that is, the probability that the particular antibody binds to an accurate antigen epitope.

The term "valence", as used herein, refers to the number of potential target binding sites in a polypeptide Each target binding site specifically binds to a target molecule or to a specific site (i.e., to an epitope) ) on a target molecule When a polypeptide comprises more than one target binding site, each target binding site can bind specifically to the same or different molecules (eg, it can bind to different molecules, eg, to different antigens). , or to different epitopes on the same molecule).

The phrase "inhibitory antibody", as used herein, refers to an antibody that binds to HER3 and inhibits The biological activity of H ER3 signaling, for example, reduces, decreases and / or inhibits the signaling activity induced by HER3, for example, in a phospho-HER3 or phospho-Akt assay. The examples of the tests are described in more detail in the Examples that follow later. In accordance with the foregoing, it will be understood that an antibody that "inhibits" one or more of these functional properties of HER3 (eg, biochemical, immunochemical, cellular, physiological, or other biological activities, or the like), as determined from According to the methodologies known in this field and described herein, it is related to a statistically significant decrease in the particular activity relative to that seen in the absence of the antibody (e.g., or when a control antibody of an irrelevant specificity is present). An antibody that inhibits the activity of HER3 effects said statistically significant decrease by at least 10 percent of the parameter measured, by at least 50 percent, 80 percent or 90 percent, and in certain modalities, an antibody of The invention can inhibit more than 95 percent, 98 percent or 99 percent of the functional activity of HER3, as evidenced by a reduction in the level of cellular phosphorylation of HER3.

The phrase "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an antibody isolate that binds specifically to HER3 is substantially free of antibodies that specifically bind antigens other than HER3). An isolated antibody that binds specifically to HER3, however, may have cross-reactivity with other antigens. Moreover, an isolated antibody can be substantially free of other cellular materials and / or chemicals.

The phrase "conservatively modified variant" applies to sequences of both amino acids and nucleic acids. With respect to particular nucleic acid sequences, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the GCA, GCC, GCG and GCU codons all encode the amino acid alanine. Accordingly, in any position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. These variations of nucleic acids are "silent variations", which are a kind of conservatively modified variations.Any nucleic acid sequence herein that encodes a polypeptide, also describes any possible variation silent nucleic acid. One skilled person will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to provide a functionally identical molecule. In accordance with the above, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

For polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions or additions to a polypeptide sequence which results in the substitution of an amino acid with a chemically similar amino acid. The tables of conservative substitutions that provide functionally similar amino acids are well known in the art. The conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, the interspecies homologs, and the alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)). In some modalities, the term "Conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

The terms "cross-competition" and "cross-competition" are used interchangeably herein to mean the ability of an antibody or a fragment thereof to interfere with the binding of other antibodies or fragments thereof to HER3 in an assay of conventional competitive link.

The ability or degree to which an antibody or a fragment thereof is capable of interfering with the binding of another antibody or fragment thereof to HER3 and, therefore, if it can be said that it competes cross-wise according to the invention, it can be determined using conventional competitive bonding tests. A suitable trial involves the use of Biacore technology (for example, using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the degree of interactions using surface plasmon resonance technology. Another trial to measure cross competition uses an ELISA-based approach.

The term "optimized", as used herein, refers to a nucleotide sequence that has been altered to encode an amino acid sequence using the codons that are preferred in the producing cell or organism, generally speaking a eukaryotic cell, for example, a Pichia cell, a Trichoderma cell, a Chinese hamster ovary cell (CHO), or a human cell. The optimized nucleotide sequence is designed to fully preserve, or as much as possible, the amino acid sequence originally encoded by the initial nucleotide sequence, which is also known as the "parent" sequence.

Conventional assays to evaluate the binding capacity of antibodies to HER3 from different species are known in the art, including, for example, ELISAs, Western blots, and RIAs. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies can also be assessed by conventional assays known in the art, such as by Biacore analysis or the relative affinity of FACS (Scatchard). Assays to evaluate the effects of antibodies on the functional properties of HER3 (e.g., receptor binding assays or modulation of the HER3 signaling pathway) are described in greater detail in the Examples.

The phrases "identical percent" or "percent identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same.Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid or nucleotide residues that are equal (ie, 60 percent identity, optionally 65 percent, 70 percent, 75 percent percent, 80 percent, 85 percent, 90 percent, 95 percent, or 99 percent identity over a specified region, or, when not specified, over the entire sequence), when they are compared and aligned for a maximum correspondence on a comparison window, or a region designated as being measured using one of the following sequence comparison algorithms, or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a reference sequence, with which the test sequences are compared. When a sequence comparison algorithm is used, the test and reference sequences are entered into a computer, the coordinates of the subsequence are designated, if necessary, and the program parameters of sequence algorithms are designated. You can use the default program parameters, or you can designate alternative parameters. Then the sequence comparison algorithm calculates the percentage of sequence identity for the test sequences relative to the reference sequence, based on the parameters of the program.

A "comparison window", as used herein, includes reference to a segment of any of the number of contiguous positions selected from the group consisting of 20 to 600, usually from about 50 to about 200, more usually from about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of contiguous positions after that the two sequences are aligned in an optimal way. Methods of alignment of the sequences for comparison are well known in the art. Optimal alignment of the sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2: 482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444, through the computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by manual alignment and visual inspection (see, for example, Brent et al. (2003) Current Protocols in Molecular Biology).

Two examples of algorithms that are suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25: 3389-3402; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. The software to carry out the analyzes BLAST is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying the high-scoring sequence pairs (HSPs), by identifying short words of a length W in the requested sequence, which agree or satisfy some threshold value of positive value T when they are aligned with a word of the same length in a sequence of the database. T is referred to as the scoring threshold of the neighboring word (Altschul et al., Supra). These initial impacts of the neighboring word act as seeds to initiate searches in order to find longer high-score sequences (HSPs) that contain them. Word hits extend in both directions along each sequence so that the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, parameters M (reward score for a pair of matching residues, always> 0), and N (penalty score for residues that do not match, always < 0 ). For the amino acid sequences, a scoring matrix is used in order to calculate the cumulative score. The extent of word hits in each direction stops when: the cumulative alignment score falls out by the amount X from its maximum reached value; the cumulative score reaches zero or less, due to the accumulation of one or more alignments of scoring residuals negative; or the end of any sequence is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 1 1, an expectation (E) of 10, M = 5, N = -4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses by default a word length of 3, and expectation (E) of 10, and the scoring matrix BLOSUM62 (see Henikoff and Henikoff, (1989) Proc. Nati. Acad. Sci. USA 89: 10915), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4, and a comparison of both chains.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Nati, Acad. Sci. USA 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The percentage of identity between two sequences of amino acids can also be determined using the algorithm of E. Mcyers and W. Miller ((1988) Comput. Appl. Biosci. 4: 1 1-17), which has been incorporated into the ALIGN program (version 2.0), using a waste weight table PAM 120, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (1970, J. Mol. Biol. : 444-453), which has been incorporated into the Gap program of the GCG software package (available at www.gcg .com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16 , 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

Other than the percentage of sequence identity mentioned above, another indication that two nucleic acid sequences or polypeptides are substantially identical, is that the polypeptide encoded by the first nucleic acid cross-reacts immunologically with the reproduced antibodies against the polypeptide encoded by the second nucleic acid, as described below. Accordingly, a polypeptide is typically substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that the two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize with each other under constraining conditions, as described in FIG. continuation . Yet another indication that the two nucleic acid sequences are substantially identical, is that the same primers can be used to amplify the sequence.

The phrase "nucleic acid" is used herein interchangeably with the term "polynucleotide", and refers to deoxyribonucleotides or ribonucleotides and polymers thereof, either in a single chain or double chain form. The term encompasses nucleic acids containing known nucleotide analogs or modified base structure residues or bonds, which are synthetic, occur naturally, and occur not naturally, which have similar binding properties to those of the reference nucleic acid , and that are etabolized in a similar way to the reference nucleotides. Examples of these analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs).

Unless indicated otherwise, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions), and complementary sequences, as well as the explicitly indicated sequence. In a specific manner, as detailed below, degenerate codon substitutions can be achieved by generating sequences where the third position of one or more (or all) selected codons are substituted with mixed base and / or deoxy-inosine residues (Batzer et al. (1991), Nucleic Acid Res. 19: 5081, Ohtsuka et al. (1985) J. Biol. Chem. 260 : 2605-2608; and Rossolini et al. (1994) Mol. Cell. Probes 8: 91-98).

The phrase "operably linked" refers to a functional relationship between two or more segments of polynucleotides (e.g., DNA). Typically, it refers to the functional relationship of a transcription regulatory sequence with a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or in another expression system. Generally speaking, transcriptional regulatory sequences of the promoter that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, ie they are of c / s action. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically contiguous or located in close proximity to the coding sequences whose transcription enhances.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.The terms apply to polymers of amino acids wherein one or more amino acid residues are an artificial chemical mimic of an amino acid That is present naturally corresponding, as well as naturally occurring amino acid polymers and amino acid polymers that do not occur naturally. Unless indicated otherwise, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

The term "subject", as used herein, includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Except when noted, the terms "patient" or "subject" are used interchangeably herein.

The term "anticancer agent", as used herein, refers to any agent that can be used to treat a cell proliferative disorder, such as cancer, including cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, agents directed against cancers, and immunotherapeutic agents.

The term "tumor", as used herein, refers to the growth and proliferation of neoplastic cells, either malignant or benign, and to all pre-cancerous and cancerous cells and tissues.

The term "anti-tumor activity", as used herein, refers to a reduction in the rate of proliferation, viability, or metastatic activity of tumor cells. A Possible way of showing the anti-tumor activity is when a decline in the growth rate of the abnormal cells is shown, which occurs during therapy, or the stability or reduction in tumor size. This activity can be assessed using accepted in vitro or live tumor models, including, but not limited to, xenomatic models, MMTV models, and other models known in the art to investigate anti-tumor activity.

The term "malignancy", as used herein, refers to a non-benign tumor or cancer.

The term "cancer", as used herein, refers to a malignancy characterized by poorly regulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas. The term "cancer" includes primary malignancies (eg, those whose cells have not migrated to the subject's body sites other than the original tumor site), and secondary malignancies (eg, those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).

Different aspects of the invention are described in greater detail in the following sections and subsections.

Structure and Mechanism of Action of HER Receptors The four HER receptors have an extracellular ligand binding domain, a single transmembrane domain, and a domain containing cytoplasmic tyrosine kinase. The intracellular tyrosine kinase domain of H ER receptors is highly conserved, although the HER3 kinase domain contains critical amino acid substitutions and, therefore, lacks kinase activity (Guy et al. (1994): PNAS 91, 8132 -8136). HER receptor dimerization induced by the ligand induces kinase action, receptor transphosphorylation on the tyrosine residues in the C-terminal tail, followed by recruitment and action of intracellular signaling effectors (Yarden and Sliwkowski , (2001) Nature Rev 2, 127-137; Jorissen et al. (2003) Exp Cell Res 284, 31 -53.

The crystal structures of the extracellular domains of the HERs have provided some insight into the process of receptor action induced by the ligand (Schlessinger, (2002) Cell 1 10, 669-672). The extracellular domain of each HER receptor consists of four subdomains: Subdomains I and III cooperate in the formation of the ligand binding site, while subdomain II (and perhaps also the IV subdomain) participates in the dimerization of the receptor by means of of direct receptor-receptor interactions. In structures of ligand-bound H ER1, a b-hairpin (referred to as the dimerization cycle) in subdomain II interacts with the dimerization cycle of the companion receptor, mediating receptor dimerization (Garrett et al. (2002) Cell 1 10, 763-773; Ogiso et al. (2002) Cell 1 10, 775-787). In contrast, in the structures of HER1, HER3 and HER4 inactive, the dimerization cycle deals with intramolecular interactions with subdomain IV, which prevents receptor dimerization in the absence of the ligand (Cho and Leahy, (2002) Science 297, 1330-1333; Ferguson et al. collaborators (2003) Mol Cell 12, 541-552; Bouyan et al. (2005) PNAS102, 15024-15029). The structure of HER2 is unique among HERs. In the absence of a ligand, HER2 has a conformation resembling the state activated by the HER1 ligand with a protruding dimerization cycle, available to interact with other HER receptors (Cho et al. (2003) Nature 421, 756-760; Garrett et al. (2003) Mol Cell 1 1, 495-505). This may explain the better heterodimerization capacity of HER2.

Although the crystal structures of the HER receptor provide a model for homo- and hetero-dimerization of the HER receptor, the background for the prevalence of some homo- and hetero-dimers of HER over others (Franklin et al. (2004) Cancer Cell 5 , 317-328), as well as the conformational function of each domain in the dimerization and self-inhibition of the receptor (Burgess et al. (2003) Mol Cell 12, 541-552; Mattoon et al. (2004) PNAS 101, 923-928 ) remains unclear. As described below, the structure of the X-ray crystal of HER3 provides more insights.

Structure of HER3 and Epitopes Previously, a conformational epitope to which the anti-HER3 antibodies are linked has been described in the Publication International TCP Number PCT / EP201 1/064407 and in the United States of America Patent Application Serial Number 61 / 375,408, both filed August 22, 201 1, and which are incorporated herein by reference In its whole. The tridimensional structure of a truncated form of HER3 that forms a complex with an antibody fragment of HER3, showed the conformational epitope comprising domain 2 and domain 4 of HER3.

The present invention provides an additional class of antibodies or fragments thereof that bind to a linear, nonlinear, or conformational epitope within domain 2 of HER3. These antibodies or fragments thereof interact with HER3, to inhibit signal transduction both ligand-dependent and ligand-independent.

In order to examine the crystal structure of domain 2 antibodies or fragments thereof linked to HER3, HER3 crystals can be prepared by expressing a nucleotide sequence encoding HER3 or a variant thereof in a suitable host cell, and then the purified proteins are crystallized, in the presence of the Fab directed by the relevant HER3. Preferably, the HER3 polypeptide contains the extracellular domain (amino acids 20 to 640 of the human polypeptide (SEQ ID NO: 1) or of a truncated version thereof, preferably comprising amino acids 20-640) but lacking the domains transmembrane and intracellular.

HER3 polypeptides can also be produced as fusion proteins, for example, to aid in extraction and purification. Examples of the components of the fusion protein include glutathione-S-transferase (GST), histidine (HIS), hexahistidine (6HIS), GAL4 (DNA binding domains and / or transcription activation domains), and beta-galactosidase . It may also be convenient to include a proteolytic cleavage site between the fusion protein component and the sequence of the protein of interest to allow the removal of the fusion protein sequences.

After expression, the proteins can be purified and / or concentrated, for example, by immobilized metal affinity chromatography, ion exchange chromatography, and / or gel filtration.

The proteins can be crystallized using the techniques described herein. Commonly, in a crystallization process, a drop containing the protein solution is mixed with the crystallization regulator, and left to equilibrate in a sealed container. Balancing can be achieved by known techniques, such as the "drop drop" or "drop drop" method. In these methods, the droplet hangs above or sits on one side of a much larger reservoir of crystallization regulator, and equilibrium is achieved through vapor diffusion. In an alternative way, the equilibration can be present by other methods, for example, under oil, through a semi-permeable membrane, or by diffusion without interface (see, for example, Chayen et al. (2008) Nature Methods 5, 147-153).

Once the crystals have been obtained, the structure can be resolved by known X-ray diffraction techniques. Many techniques use chemically modified crystals, such as those modified by heavy atom derivation up to the approximate phases. In practice, a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, for example, lead chloride, gold thiomalate, thimerosal, or uranyl acetate, which can diffuse through the crystal and they bind to the surface of the protein. The locations of the linked heavy metal atoms can then be determined by X-ray diffraction analysis of the soaked glass. The patterns obtained in the diffraction of a monochromatic beam of X-rays by means of the atoms (dispersion centers) of the crystal can be solved by means of mathematical equations to give the mathematical coordinates. The diffraction data is used to calculate an electron density map of the crystal repeat unit. Another method to obtain phase information is by employing a technique known as molecular replacement. In this method, rotation and translation algorithms are applied to a derived search model from a related structure, which results in an approximate orientation for the protein of interest (see Rossmann, (1990) Acta Crystals A 46, 73-82). Electron density maps are used to establish the positions of individual atoms within the unit cell of the crystal (Blundel et al. (1976) Protein Crystallography, Academic Press).

The approximate domain boundaries of the extracellular domain of HER3 are as follows; domain 1: amino acids 20-207; domain 2: amino acids 208-328; domain 3: amino acids 329-498; and domain 4: amino acids 499-642. The three-dimensional structure of HER3 and the antibody allows the identification of the target binding sites for the potential modulators of HER3. Preferred target binding sites are those involved in the activation of HER3. In one embodiment, the target link site is located within domain 3 of HER3. Accordingly, an antibody or a fragment thereof that binds to domain 3, for example, can modulate HER3 activation by modifying the relative position of the domain in relation to itself or to other HER3 domains. Accordingly, binding of an antibody or a fragment thereof to the amino acid residues within domain 3 may cause the protein to adopt a configuration that prevents activation or prevents dimerization with dimerizing components (e.g., H ER2). ).

In some embodiments, the antibody or a fragment thereof recognizes a specific conformational state of HER3, such that the antibody or a fragment thereof prevents HER3 from interacting with a co-receptor (including, but not limited to, HER1, HER2 and HER4). In some embodiments, the antibody or a fragment thereof prevents HER3 from interacting with a co-receptor by stabilizing the HER3 receptor in an inactive or closed state. In some embodiments, the antibody or a fragment thereof can stabilize the HER3 receptor by binding to amino acid residues within domain 2 of HER3. In some embodiments, the antibody or a fragment thereof binds to the human HER3 protein having an epitope comprising the amino acid residues of HER3 within domain 2 (amino acids 208-328 of SEQ ID NO: 1), or a subset of them. In some embodiments, the antibody or a fragment thereof binds to the amino acids within, or overlapped with, the amino acid residue within domain 2 (amino acids 208-328 of SEQ ID NO: 1). The antibody or a fragment thereof described herein can be linked to Lys 268 within domain 2 of HER3. In some embodiments, the antibody or fragment thereof binds to a linear epitope within domain 2 of HER3. In some embodiments, the antibody or a fragment thereof binds to a non-linear epitope within domain 2 of HER3. In some embodiments, the antibody or a fragment thereof binds to a conformational epitope within domain 2 of HER3.

In some embodiments, the antibody or a fragment thereof binds to the epitope in domain 2 of HER3, thereby that the dimerization cycle within domain 2 of HER3 is not available for dimerization with a co-receptor. Failure to form homo- or hetero-dimers results in failure to activate signal transduction.

In some embodiments, the antibody or a fragment thereof can bind to an epitope in domain 2 either in the active or inactive state of HER3.

In some embodiments, the antibody or a fragment thereof binds to an epitope in domain 2 of the HER3 receptor, wherein binding of the antibody or a fragment thereof to the HER3 receptor allows dimerization with a co-receptor to form a inactive receptor-receptor complex. The formation of the inactive receptor-receptor complex prevents the activation of signal transduction independent of the ligand. For example, in signal transduction independent of the ligand, HER3 can exist in an inactive state; however, over-expression of HER2 causes the formation of the HER2-HER3 complex; however, these resulting complexes are inactive and prevent the activation of signal transduction independent of the ligand.

In some embodiments, domains / regions containing residues that are in contact with, or that are buried by, an antibody can be identified by mutation of specific residues in HER3 (eg, a wild-type antigen), and by determining whether the antibody or fragment thereof can bind to the mutated protein or HER3 variant, or by measuring the affinity changes from the wild type. By making a number of individual mutations, residues having a direct function in the link, or which are in a proximity sufficiently close to the antibody, can be identified such that a reaction can affect the bond between the antibody and the antigen. From a knowledge of these amino acids, domains or regions of the antigen (HER3) containing residues in contact with the antibody, or that are covered by the antibody, can be elucidated. Mutagenesis using known techniques, such as the alanine scan, can help define functionally relevant epitopes. Mutagenesis can also be employed using an arginine / glutamic acid scanning protocol (see, for example, Nanevicz et al. (1995), J. Biol. Chem. 270 (37): 21619-21625 and Zupnick et al. (2006). , J. Biol. Chem. 281 (29): 20464-20473). In general, arginine and glutamic acids are used to replace (typically in an individual manner) an amino acid in the wild-type polypeptide, because these amino acids are charged and bulky and, therefore, have the potential to disrupt the binding between an antigen binding protein and an antigen in the region of the antigen where the mutation is introduced. The arginines that exist in the wild-type antigen are replaced with glutamic acid. A variety of these individual mutants can be obtained, and the binding results collected can be analyzed to determine which residues affect the link. A series of mutant HER3 antigens can be created, each mutant antigen having a single mutation. The binding of each mutant HER3 antigen with different antibodies or fragments thereof of HER3, can be measured and compared with the ability of the selected antibody or fragments thereof to bind to the wild type HER3 (SEQ ID NO: 1 ). Examples of these mutants are shown later in the Examples section, for example, the mutant of Ais Lys 268.

An alteration (eg, a reduction or an increase) in the link between an antibody or a fragment thereof and a mutant or variant HER3, as used herein, means that there is a change in binding affinity (eg, as measured by known methods, such as the Biacore test, or the granule-based assay described later in the Examples), in the EC50, and / or a change (e.g., a reduction) in the total binding capacity of the antigen-binding protein (e.g., as evidenced by a decrease in Bmax in a protein concentration plot of link to the antigen against the concentration of the antigen). A significant alteration in the linkage indicates that the mutated residue is involved in the binding to the antibody or fragment thereof.

In some embodiments, a significant reduction in binding means that the binding affinity, the EC50, and / or the capacity between an antibody or fragments thereof and an antigen of HER3 mutant, is reduced by more than 10 percent, by more than 20 percent, by more than 40 percent, by more than 50 percent, by more than 55 percent, by more than 60 percent, by more than 65 percent, by more than 70 percent, by more than 75 percent, by more than 80 percent, by more than 85 percent, by more than 90 percent or by more than 95 percent in relation to the link between the antibody or a fragment thereof and a wild-type HER3 (eg, SEQ ID NO: 1).

In some embodiments, the binding of an antibody or fragments thereof is reduced or significantly increased for a mutant protein of HER3 having one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mutations, compared to a wild type protein of HER3 (eg, SEQ ID NO: 1).

Although the variant forms are referenced with respect to the wild-type sequence shown in SEQ ID NO: 1, it will be appreciated that in the allelic or splicing variants of HER3 the amino acids may be different. Antibodies or fragments thereof that show a significantly altered linkage (e.g., a lower or higher binding) for those allelic forms of HER3 are also contemplated.

In addition to the general structural aspects of the antibodies, the more specific interaction between the paratope and the epitope can be examined through structural approaches. In one modality, the structure of the determining regions of Complementarity contributes to a paratope, through which an antibody is able to bind to an epitope. The shape of this paratope can be determined in a number of ways. Traditional approaches to structural examination, such as MRI or X-ray crystallography, can be used. These approaches can examine the shape of the paratope alone, or while linking to the epitope. In an alternative way, in silico molecular models can be generated. A structure can be generated through homology modeling, aided by a commercial package, such as the Insightll modeling package from Accelrys (San Diego, Calif.). Briefly stated, the sequence of the antibody to be examined can be used to search against a database of proteins of known structures, such as the Protein Data Bank. After identifying the homologous proteins with known structures, these homologous proteins are used as the modeling templates. Each of the possible templates can be aligned, therefore, producing sequence alignments based on the structure between the templates. The sequence of the antibody with the unknown structure can then be aligned with these templates to generate a molecular model for the antibody with the unknown structure. As will be appreciated by one skilled in the art, there are many alternative methods for generating these in silico structures, any of which can be used. For example, a process similar to that described in Hardman and co-workers, U.S. Pat. No. 5,958,708 issued to QUANTA (Poligen Corp., Waltham, Mass.), and CHARM (Brooks et al. (1983), J. Comp. Chem. 4: 187) (incorporated herein). present in its entirety as a reference).

Not only is the shape of the paratope important in determining if and how well a possible paratope will bind to an epitope, but the very interaction between the epitope and the paratope is a source of great information in the design of the variant antibodies. As appreciated by one skilled in the art, there are a variety of ways in which this interaction can be studied. One way is to use the structural model generated, perhaps as described above, and then use a program, such as Insightll (Accelrys, San Diego, Calif.), which has a docking module, which, among other things, is capable of carrying out a search Monte Carlo on the conformational and orientation spaces between the paratope and its epitope. The result is that you can estimate where and how the epitope interacts with the paratope. In one embodiment, only one fragment or one variant of the epitope is used to assist in the determination of the relevant interactions. In one embodiment, the whole epitope is used in the modeling of the interaction between the paratope and the epitope.

Through the use of these modeled structures, one can predict which residues are the most important in the interaction between the epitope and the paratope. Accordingly, in one embodiment, one can easily select which residues to change in order to alter the binding characteristics of the antibody. For example, it may be evident, from mooring models, that the side chains of certain residues in the paratope can sterically prevent the binding of the epitope, and therefore, it may be beneficial to alter these residues until there are residues with side chains more little. You can determine this in many ways. For example, one can simply look at the two models and estimate the interactions based on the functional groups and proximity. Alternatively, repeated epitope and paratope pairings can be carried out, as described above, in order to obtain more favorable energy interactions. These interactions can also be determined for a variety of antibody variants in order to determine alternative ways in which the antibody can bind to the epitope. The different models can also be combined to determine how the structure of the antibodies should be altered in order to obtain an antibody with the particular characteristics that are desired.

The models determined above can be tested through different techniques. For example, the interaction energy can be determined with the programs discussed above in order to determine which of the variants will be examined Additionally. Also, coulúmbic and van der Waals interactions are used to determine the interaction energies of the epitope and the variant paratopes. Site-directed mutagenesis is also used to see if the predicted changes in antibody structure actually result in the desired changes in binding characteristics. Alternatively, changes can be made to the epitope in order to verify that the models are correct or to determine the general linkage issues that may arise between the paratope and the epitope.

As will be appreciated by one skilled in the art, although these models will provide the necessary guidance for making the antibodies and variants thereof of the present embodiments, it may still be desirable to carry out the routine tests of the in silico models, such once through in vitro studies. In addition, as will be apparent to one skilled in the art, any modification may also have additional side effects on the activity of the antibody. For example, although any alteration predicted to result in a greater binding may induce a greater binding, it may also cause other structural changes that could reduce or alter the activity of the antibody. The determination of whether this is the case or not is routine in the matter and can be achieved in many ways. For example, the activity can be tested through an ELISA test. Alternatively, samples can be tested through the use of a surface plasmon resonance apparatus.

Antibodies of HER3 The present invention provides antibodies that recognize an epitope within domain 2 of HER3. The invention is based on the surprising finding that a class of antibodies against HER3, block the HER3 signal transduction pathways both ligand-dependent and ligand-independent in Table 1, discloses a class of antibodies that bind to an epitope within domain 2 of HER3. In one embodiment, the antibodies inhibit HER3 signaling both ligand-dependent and ligand-independent. In another embodiment, the antibodies bind to HER3 and do not block the binding of the HER ligand to the ligand binding site (ie, both the ligand and the antibody can bind to HER3 in a concurrent manner).

Table 1 Examples of HER3 antibodies that bind to a domain 2 of HER3 The present invention provides antibodies that specifically bind to a HER3 protein (eg, human and / or cynomolgus HER3), the antibodies comprising a VH domain having an amino acid sequence of SEQ ID NOs: 14, 34, 54 , 74, 94, 1 14, 134, 154, 174, 194, 214, 234, 254, 274, 294, 314, 334, 354, 374, 394, 414, 434, 454, 474, 494, 514, and 524 .

The present invention provides antibodies that bind specifically with a HER3 protein (eg, human HER3 and / or cynomolgus), the antibodies comprising a VL domain having an amino acid sequence of SEQ ID NOs: 15, 35, 55, 75, 95, 1 15, 135, 155, 175, 195, 215, 235, 255, 275, 295, 315, 335, 355, 375, 395, 415, 435, 455, 475, 495, 515, and 535.

The present invention also provides antibodies that specifically bind to a HER3 protein (eg, human and / or cynomolgus HER3), these antibodies comprising a VH CDR having an amino acid sequence of any of the VH CDRs listed in the Table. 1. In particular, the invention provides antibodies that specifically bind to a HER3 protein (e.g., human and / or cynomolgus HER3), these antibodies comprising (or alternatively, consisting of) one, two, three, four, five or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 1.

Other antibodies of the invention include amino acids that have mutated, and yet have at least 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent one hundred, 98 percent or 99 percent identity in the complementarity determining regions (CDRs) with the complementarity determining regions (CDRs) illustrated in the sequences described in Table 1. In some embodiments, it includes mutant amino acid sequences where no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the complementarity determining regions (CDRs) when compared to the complementarity determining regions (CDRs) illustrated in the sequence described in Table 1, while still maintaining their specificity for the epitope of the original antibody Other antibodies of the invention include amino acids that have mutated, and yet have at least 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent percent, 98 percent or 99 percent identity in the structure regions with the structure regions illustrated in the sequences described in Table 1. In some embodiments, includes mutant amino acid sequences wherein no more than 1, 2, 3, 4, 5, 6, or 7 amino acids have been mutated in the structure regions when compared to the structure regions illustrated in the sequence described in the Table 1, while still maintaining its specificity for the epitope of the original antibody. The present invention also provides nucleic acid sequences encoding VH, VL, the full-length heavy chain, and the full-length light chain of antibodies that specifically bind to a HER3 protein (e.g., human and / or HER3). of cinomolgo).

Other antibodies of the invention include those wherein the amino acids or nucleic acids encoding the amino acids have been mutated, and yet they have at least 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent identity with the sequences described in Table 1. In some embodiments, they include mutant amino acid sequences where no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable regions when compared to the variable regions illustrated in the sequence described in Table 1, while retaining substantially the same therapeutic activity.

Because each of these antibodies or fragments thereof can be linked to HER3, the full-length light chain, VH, VL, and heavy chain sequences (the amino acid sequences and nucleotide sequences encoding the amino acid sequences) can be "mixed and matched" to create other HER3 antibodies of the invention. These "mixed and matched" antibodies of HER3 can be tested using binding assays known in the art (eg, ELISAs, and other assays described in the Examples section). When these chains are mixed and matched, a VH sequence from a particular VH / VL pairing must be replaced with a structurally similar VH sequence. In the same manner, a full-length heavy chain sequence from a particular full length heavy chain / full length light chain match must be replaced with a structurally similar full length heavy chain sequence. In the same way, a VL sequence from a pairing Particular VH / VL must be replaced with a structurally similar VL sequence. In the same manner, a full length light chain sequence from a particular full length heavy chain / light chain full length match must be replaced with a structurally similar full length light chain sequence.

In accordance with the above, in one aspect, the invention provides an isolated monoclonal antibody or a fragment thereof having: VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54 , 74, 94, 1 14, 134, 154, 174, 194, 214, 234, 254, 274, 294, 314, 334, 354, 374, 394, 414, 434, 454, 474, 494, 514, and 524; and VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 35, 55, 75, 95, 1 15, 135, 155, 175, 195, 215, 235, 255, 275, 295, 315, 335, 355, 375, 395, 415, 435, 455, 475, 495, 515, and 535; wherein the antibody binds specifically to HER3 (eg, human and / or cynomolgus).

In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 14 and a VL of SEQ ID NO: 15. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 34 and a VL of SEQ ID NO: 35. In a specific embodiment, an antibody that binds HER3 comprises a VH of SEQ ID NO: 54 and a VL of SEQ ID NO: 55. In a specific modality, an antibody which binds to H ER3 comprises SEQ ID NO: 74 and a VL of SEQ ID NO: 75. In a specific embodiment, an antibody that binds HER3 comprises a VH of SEQ ID NO: 94 and a VL of SEQ ID NO: 95. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 1 14 and a VL of SEQ ID NO: 1 15. In a specific embodiment, an antibody that is linked to HER3 comprises a VH of SEQ ID NO: 134 and a VL of SEQ ID NO: 135. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 154 and a VL of SEQ ID NO: 155. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 174 and a VL of SEQ ID NO: 175. In a specific embodiment, an antibody that is link to HER3 comprises a VH of SEQ ID NO: 194 and a VL of SEQ ID NO: 195. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 214 and a VL of SEQ ID NO: 215. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 234 and a VL of SEQ ID NO: 235. In a specific embodiment, an antibody that binds to H ER3 comprises a VH of SEQ ID NO: 254 and a VL of SEQ ID NO: 255. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 274 and a VL of SEQ ID NO: 275. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 294 and a VL of SEQ ID NO: 295. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 314 and a VL of SEQ ID NO: 315. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 334 and a VL of SEQ ID NO: 335. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 354 and a VL of SEQ ID NO: 355. In an embodiment specifically, an antibody that binds to H ER3 comprises a VH of SEQ ID NO: 374 and a VL of SEQ ID NO: 375. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 394 and a VL of SEQ ID NO: 395. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 414 and a VL of SEQ ID NO: 415. specific, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 434 and a VL of SEQ ID NO: 435. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 454 and a VL of SEQ ID NO: 455. In a specific embodiment, an antibody that binds to HER3 comprises a VH of the SEQ ID NO: 474 and a VL of SEQ ID NO: 475. In a specific embodiment, an antibody that binds HER3 comprises a VH of SEQ ID NO: 494 and a VL of SEQ ID NO: 495. a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 514 and a VL of SEQ ID NO: 515. In a specific embodiment, an antibody that binds to HER3 comprises a VH of SEQ ID NO: 534 and one VL of SEQ ID NO: 535.

In another aspect, the present invention provides HER3 antibodies comprising the heavy chain and light chain of the CDR1 s, CDR2s and CDR3s as described in Table 1, or combinations thereof. The complementarity determining regions (CDRs) are delineated using the Kabat system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Qumta Edition, U.S. Department of Health and Human Services (Department of Health and Human Services). the United States), N 1H Publication No. 91-3242, Chotia et al. (1987) J. Mol. Biol. 196: 901-917; Chotia et al. (1989) Nature 342: 877-883; and Al-Lazikani and collaborators (1997) J. Mol. Biol. 273, 927-948). In accordance with the foregoing, in one embodiment, the antibody or fragment thereof comprises a heavy chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 2, 22 , 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, and 522; a sequence of CDR2 selected from the group consisting of SEQ ID NOs: 3, 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323 , 343, 363, 383, 403, 423, 443, 463, 483, 503, and 523; and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, and 524; and a light chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, and 528; a sequence of CDR2 selected from the group consisting of SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, 249, 269, 289, 309, 329 , 349, 369, 389, 409, 429, 449, 469, 489, 509, and 529; and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290 , 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, and 530, wherein the antibody or a fragment thereof binds to domain 2 of HER3.

In a specific embodiment, an antibody that binds to HER3 comprises a heavy chain variable region CDR1 of SEQ ID NO: 502; a CDR2 of SEQ ID NO: 503; a CDR3 of SEQ ID NO: 504; a light chain variable region CDR1 of SEQ ID NO: 508; a CDR2 of SEQ ID NO: 509; and a CDR3 of SEQ ID NO: 510.

In a specific embodiment, an antibody that binds HER3 comprises a heavy chain variable region CDR1 of SEQ ID NO: 522; a CDR2 of SEQ ID NO: 523; a CDR3 of SEQ ID NO: 524; a light chain variable region CDR1 of SEQ ID NO: 528; a CDR2 of SEQ ID NO: 529; and a CDR3 of SEQ ID NO: 530.

As used herein, a human antibody comprises heavy or light chain variable regions or full-length heavy or light chains that are "the product of" or "derived from" a particular germline sequence if variable regions or full-length chains of the antibody are obtained from a system using human germline immunoglobulin genes. These systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest, or screening a library of human immunoglobulin genes displayed on the phage with the antigen of interest. A human antibody that is "the product of" or "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of the immunoglobulins of the human human germline, and selecting the human germline immunoglobulin sequence that is closest in sequence (ie, highest percent identity) to the human antibody sequence. A human antibody that is "the product of" or "derived from" a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, due, for example, to mutations somatic diseases that occur naturally or the intentional introduction of mutations directed to the site. However, in VH or VL structure regions, a human antibody typically selected is at least 90 percent identical in amino acid sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody human as compared to the germline immunoglobulin amino acid sequences of other species (eg, murine germ line sequences). In certain cases, a human antibody can be at least 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent identical in the amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a recombinant human antibody will exhibit no more than 10 amino acid differences of the amino acid sequence encoded by the human germline immunoglobulin gene in regions of structure VH or VL. In certain cases, the human antibody can exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference of the amino acid sequence encoded by the germline immunoglobulin gene.

The antibodies disclosed herein may be derived from single chain antibodies, diabodies, domain antibodies, nanobodies, and antibodies. A "single chain antibody" (scFv) consists of a single chain of polypeptide comprising a VL domain linked to a VH domain, wherein the VL domain and the VH domain are paired to form a monovalent molecule. The single chain antibody can be prepared according to the method known in the art (see, for example, Bird et al. (1988) Science 242: 423-426, and Huston et al. (1988) Proc. Nati Acad. Sci. USA 85: 5879-5883). A "disbud" consists of two chains, each chain comprising a variable region of heavy chain connected to a variable region of light chain on the same chain of the polypeptide, connected by a short peptide linker, where the two regions on the same chain do not they pair with each other, but with the complementary domains on the other chain, to form a bispecific molecule. Methods for the preparation of diabodies are known in the art (see, for example, Holliger et al. (1993) Proc. Nati, Acad. Sci. USA 90: 6444-6448, and Poljak et al. (1994) Structure 2: 1 121-1 123). The domain antibodies (dAbs) are small units of functional binding antibodies, corresponding to the variable regions of either the heavy or light chains of the antibodies. Domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Other details of the domain antibodies and production methods thereof are known in the art (see, for example, US Patents Numbers 6,291, 158; 6,582,915; 6,593,081; 6, 172, 197; 6,696,245; the European Patent Nos. 0368684 and 0616640; and International Publications Nos. WO05 / 035572, W004 / 101790, WO04 / 081026, W004 / 058821, W004 / 003019 and W003 / 002609. The nanobodies are derived from the heavy chains of an antibody. A nanobody typically comprises a single variable domain and two constant domains (CH2 and CH3), and retains the ability to bind the antigen of the original antibody. Nanobodies can be prepared by methods known in the art (see, for example, U.S. Patent No. 6,765,087, U.S. Patent No. 6,838,254, and International Publication Number WO 06/079372) . The antibodies consist of a light chain and a heavy chain of an IgG4 antibody. The antibodies can be made by removing the region of articulation of IgG4 antibodies. Other details of the antibodies and the methods for preparing them can be found in International Publication Number W02007 / 059782.

Homologous antibodies In still another embodiment, the present invention provides an antibody or fragment thereof, which comprises the amino acid sequences that are homologous to the sequences described in Table 1, and the antibody binds to a HER3 protein (e.g., HER3 human and / or cynomolgus), and preserves the desired functional properties of the antibodies described in Table 1 .

For example, the invention provides an isolated monoclonal antibody (or a functional fragment thereof), which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 1 14, 134, 154, 174, 194, 214, 234, 254, 274, 294, 314, 334, 354, 374, 394, 414, 434, 454, 474, 494 , 514, and 524; the light chain variable region comprises an amino acid sequence that is at least 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent identical to a selected amino acid sequence from the group consisting of SEQ ID NOs: 14, 34, 54, 74, 94, 1 14, 134, 154, 174, 194, 214, 234, 254, 274, 294, 314, 334, 354, 374 , 394, 414, 434, 454, 474, 494, 514, and 524; the antibody binds to HER3 (e.g., human and / or cynomolgus HER3), and inhibits HER3 signaling activity, which can be measured in a phosphorylation assay or other HER signaling measure (e.g. phospho-HER3 assays, phospho-Akt assays, cell proliferation, and ligand blocking assays, as described in the Examples). Also included within the scope of the invention are the sequences of nucleotides progenitors of variable heavy and light chains; and the full-length heavy and light chain sequences optimized for expression in a mammalian cell. Other antibodies of the invention include amino acids or nucleic acids that have mutated, and yet have at least 60, 70, 80, 90, 95, 98, or 99 percent identity with the sequences described above. In some embodiments, mutant amino acid sequences are included wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated by deletion, insertion or substitution of amino acids in the variable regions when compared to the variable regions illustrated in the sequence described above.

In other embodiments, the VH and / or VL amino acid sequences may be 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent identical to the sequences stipulated in Table 1. In other modalities, the VH and / or VL amino acid sequences may be identical except for an amino acid substitution in no more than 1, 2, 3, 4, or 5 amino acid positions. An antibody having the VH and VL regions that have high (ie, 80 percent or greater) identity with the VH and VL regions of the antibodies described in Table 1, can be obtained by mutagenesis (e.g., site-directed mutagenesis). site or mediated by polymerase chain reaction (PCR)), followed by the altered second antibody test encoded for the function preserved using the functional assays described herein.

In other embodiments, the variable regions of the heavy chain and / or light chain nucleotide sequences may be 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent identical to the sequences stipulated above.

As used herein, the "percent identity" between the two sequences is a function of the number of identical positions shared by the sequences (i.e., the percent identity is equal to the number of identical positions / the total number of positions x 100), taking into account the number of holes, and the length of each hole, that needs to be introduced for an optimal alignment of the two sequences. The comparison of the sequences and the determination of the percentage of identity between two sequences can be carried out using a mathematical algorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the present invention can be further used as a "requested sequence" to conduct a search against public databases, for example, to identify related sequences. For example, these searches can be carried out using the BLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10.

Antibodies with conservative modifications In certain embodiments, an antibody of the invention has a heavy chain variable region comprising the sequences of CDR1, CDR2, and CDR3, and a light chain variable region comprising the sequences of CDR1, CDR2, and CDR3, wherein a or more of these sequences of complementarity determining regions (CDRs) have the amino acid sequences specified based on the antibodies described herein or on conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the HER3 antibodies. of the invention.

In accordance with the foregoing, the invention provides an isolated HER3 monoclonal antibody, or a fragment thereof, consisting of a heavy chain variable region, which comprises the sequences of CDR1, CDR2, and CDR3, and a variable region of light chain, which comprises the sequences of CDR1, CDR2, and CDR3, wherein: the amino acid sequences of the heavy chain variable region CDR1 are selected from the group consisting of SEQ ID NOs: 2, 22 , 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, and 522, and the conservative modifications thereof; the amino acid sequences of the heavy chain variable region CDR2 are selected from the group consisting of SEQ ID NOs: 3, 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, and 523, and the conservative modifications thereof; the amino acid sequences of the CDR3 heavy chain variable region are selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, and 524, and the conservative modifications thereof; the amino acid sequences of the CDR1 light chain variable regions are selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, and 528, and the conservative modifications thereof; the amino acid sequences of the CDR2 light chain variable regions are selected from the group consisting of SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, and 529, and the conservative modifications thereof; the amino acid sequences of the CDR3 light chain variable regions are selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 1 10, 130, 150, 170, 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, and 530, and the conservative modifications thereof; the antibody or a fragment thereof binds specifically to H ER3, and inhibits the activity of HER3 by inhibiting a signaling pathway of HER3 which can be measured in a phosphorylation assay or other measurement of HER signaling (eg, phospho-HER3 assays, phospho-Akt assays, cell proliferation, and ligand blocking assays, as described in the examples).

Antibodies that bind to the same epitope The present invention provides antibodies that interact (e.g., by binding, spherical hindrance, stabilization / destabilization, spatial distribution) with the same epitope as the HER3 antibodies described in Table 1. Accordingly, additional antibodies can be identified based on their ability to cross compete (for example, to competitively inhibit the binding, in a statistically significant manner) with other antibodies of the invention in HER3 binding assays. The ability of a test antibody to inhibit the binding of the antibodies of the present invention to a HER3 protein (eg, human and / or cynomolgus HER3) demonstrates that the test antibody can compete with that antibody for binding to HER3; this antibody, according to a non-limiting theory, can be linked to the same epitope or to a related epitope (eg, to a structurally similar or spatially proximal epitope) on the HER3 protein than the antibody with which it competes. In a certain embodiment, the antibody that binds to the same epitope on the HER3-related protein as the antibodies of the present invention is a human monoclonal antibody. These antibodies Human monoclonal antibodies can be prepared and isolated as described herein.

In one embodiment, the antibody or fragments thereof bind to domain 2 of HER3 to maintain HER3 in the conformation that prevents exposure of a dimerization cycle present within domain 2. This prevents heterodimerization with other members of the family , such as HER1, HER2, and HER4. Antibodies or fragments thereof inhibit HER3 signal transduction both ligand-dependent and ligand-independent.

In another embodiment, the antibody or a fragment thereof binds to domain 2 of HER3 without blocking the concurrent binding of a HER3 ligand, such as neurregulin. Although it is not required to provide a theory, it is feasible for the antibody or a fragment thereof to bind to domain 2 of HER3, and keep HER3 in a conformation that does not block the ligand binding site on HER3. Accordingly, a HER3 ligand (e.g., neurregulin) is capable of binding to HER3 at the same time as the antibody or a fragment thereof.

Antibodies of the invention or fragments thereof inhibit HER3 activation both ligand-dependent and ligand-independent without preventing linkage of the ligand. This is considered convenient for the following reasons: (i) The therapeutic antibody would have clinical utility in a broad spectrum of tumors that an antibody whose objective is a only mechanism of HER3 activation (ie, ligand-dependent or ligand-independent), because different types of tumors are handled by each mechanism. (ii) The therapeutic antibody would be effective in tumor types where the mechanisms of HER3 activation are simultaneously involved. An antibody that targets a single mechanism of HER3 activation (ie, ligand-dependent or ligand-independent) may exhibit little or no efficacy in this type of tumor. (iii) The efficacy of an antibody that inhibits HER3 ligand-dependent activation without preventing linkage of the ligand may be less likely to be adversely affected by increasing ligand concentrations. This could be translated to either an increased efficacy in a tumor type driven by very high concentrations of the ER3 H ligand, or a reduced resistance to the risk of the drug, where the resistance is mediated by the over-regulation of the ligands of HER3. (iv) An antibody that inhibits HER3 activation by stabilizing the inactive form may be less likely to have resistance to the drug, driven by alternative mechanisms of HER3 activation.

Accordingly, the antibodies of the invention can be used to treat conditions where clinically ineffective therapeutic antibodies exist.

Antibodies designed and modified An antibody of the invention can be further prepared using an antibody having one or more of the VH and / or VL sequences shown herein as a starting material for designing a modified antibody, which modified antibody can have altered properties relative to the starting antibody. An antibody can be designed by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), for example, within one or more complementarity determining regions (CDR) and / or within of one or more structure regions. Additionally or in an alternative manner, an antibody can be designed by modifying the residues within the constant regions, for example, to alter the effector functions of the antibody.

One type of variable region design that can be carried out is the graft of the complementarity determining region (CDR). The antibodies interact with the target antigens predominantly through the amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the complementarity determining regions (CDRs) are more diverse between the individual antibodies than the sequences that are outside the complementarity determining regions (CDRs). Because the sequences of complementarity determining regions (CDR) are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of antibodies that occur naturally-specific by constructing expression vectors that include sequences of complementarity determining regions (CDRs) at Starting from the naturally occurring specific antibody grafted onto the structure sequences from a different antibody with different properties (see, for example, Riechmann et al. (1998) Nature 332: 323-327; Jones et al. (1986) Nature 321 : 522-525; Queen et al. (1989) Proc. Nati. Acad., USA 86: 10029-10033; United States Patent Number 5,225,539 to Winter; and United States of America Patents 5,530; 101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In accordance with the above, another embodiment of the invention pertains to an isolated HER3 monoclonal antibody, or to a fragment thereof, which comprises a heavy chain variable region, which comprises the CDR1 sequences having a selected amino acid sequence. from the group consisting of SEQ ID NOs: 2, 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, and 522; the CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, and 523; the CDR3 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, and 524, respectively; and a light chain variable region having the CDR1 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, and 528; the CDR2 sequences having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, and 529; and the CDR3 sequences consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 1 10, 130, 150, 170, 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, and 530, respectively. Accordingly, these antibodies contain the sequences of the complementarity determining regions (CDRs) VH and VL of the monoclonal antibodies, and nevertheless, may contain different structure sequences from these antibodies. These structure sequences can be obtained from public DNA databases or from published references that include sequences of germline antibody genes. For example, the germline DNA sequences for the human and heavy chain variable region genes can be found in the human germline sequence database "Vase" (available on the Internet at www.mrc.com). - cpe.cam.ac.uk/vbase), as well as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Qumta Edition, U.S. Department of Health and Human Services, N1H Publication No. 91 -3242; Chothia et al. (1987) J. Mol. Biol. 196: 901-917; Chothia et al (1989) Nature 342: 877-883; and Al-Lazikani et al. (1997) J. Mol. Biol. 273: 927-948; Tomlinson et al. (1992) J. fol. Biol. 227: 776-798; and Cox et al. (1994) Eur. J Immunol. 24: 827-836; the content of each of which is expressly incorporated herein by reference.

An example of the structure sequences for use in the antibodies of the invention are those that are structurally similar to the structure sequences used by the antibodies selected from the invention, for example, the sequences in consensus and / or the structure sequences used. by the monoclonal antibodies of the invention. The sequences of CDR 1, 2 and 3 VH, and the sequences of CDR1, 2 and 3 VL, can be grafted onto the regions of structure that have a sequence identical to that found in the immunoglobulin gene of the line The germline from which the structure sequence is derived, or the sequences of complementarity determining regions (CDRs) can be grafted onto the structure regions containing one or more mutations compared to the germline sequences. For example, it has been found that, in certain instances, it is beneficial to mutate residues within framework regions to maintain or enhance the antigen binding capacity of the antibody (see, for example, US Pat. Nos. 5,530 101, 5,585,089, 5,693,762 and 6, 180,370 to Queen et al.).

Another type of variable region modification is to mutate the amino acid residues within the CDR1, CDR2 and / or CDR3 VH and / or VL regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, this being known as "affinity maturation." Site-directed mutagenesis or mutagenesis mediated by polymerase chain reaction (PCR) can be carried out to introduce the mutations, and the effect on the binding of the antibody, or other functional property of interest, can be evaluated in in vitro assays or in vivo, as described herein, and as provided in the Examples. Conservative modifications can be introduced (as discussed above). Mutations can be substitutions, additions or deletions of amino acids. Moreover, typically no more than one, two, three, four or five residues are disturbed within a determinant region of complementarity (CDR).

In accordance with the foregoing, in another embodiment, the invention provides isolated HER3 monoclonal antibodies, or fragments thereof, which consist of a heavy chain variable region having: a VH CDR1 region consisting of a selected amino acid sequence from the group having the SEQ ID NOs: 2, 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, and 522, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions, compared to SEQ ID NOs: 2, 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, and 522; a VH CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, and 523, or an amino acid sequence having one, two, three, four or five amino acid substitutions, deletions or additions, compared to SEQ ID NOs: 3, 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, and 523; a VH CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, and 524, or a sequence of amino acids having one, two, three, four or five substitutions, deletions or additions of amino acids, compared to SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 184, 204 , 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, and 524; a VL CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268 , 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, and 528, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids, compared to SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408 , 428, 448, 468, 488, 508, and 528; a VL CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, and 529, or an amino acid sequence having one, two, three, four or five substitutions, deletions or additions of amino acids, compared to SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, and 529; and a VL CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 1 10, 130, 150, 170, 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, and 530, or a sequence of amino acids having one, two, three, four or five substitutions, deletions or additions of amino acids, compared to SEQ ID NOs: 10, 30, 50, 70, 90, 1 10, 130, 150, 170, 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, and 530.

Grafting of antibody fragments into alternative structures or scaffolds A wide variety of antibody / immunoglobulin structures or scaffolds may be employed as long as the resulting polypeptide includes at least one binding region that specifically binds HER3. These structures or scaffolds include the 5 major idiotypes of human immunoglobulins, or fragments thereof, and include the immunoglobulins of other animal species, preferably having humanized aspects. The experts in this field continue discovering and developing structures, scaffolding and novel fragments.

In one aspect, the invention pertains to the generation of non-immunoglobulin-based antibodies using non-immunoglobulin scaffolds on which the complementarity determining regions (CDRs) of the invention can be grafted. Structures and scaffolds other than known or future immunoglobulin may be employed, provided that they comprise a specific binding region for the target HER3 protein (eg, human HER3 and / or cynomolgus). The structures or scaffolds that are not known immunoglobulin include, but are not limited to, fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge , MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immunological pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), mammalian (Avidia, Inc., Mountain View, CA ), Protein A (Affibody AG, Sweden), and afilin (gamma-crystalline or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

The fibronectin scaffolds are based on the fibronectin type III domain (for example, the tenth module of fibronectin type II I (domain 10 Fn3)). The fibronectin type III domain has 7 or 8 beta chains that are distributed between two beta sheets, which by themselves are packaged against each other to form the core of the protein, and which also contain cycles (analogous to the complementarity determining regions). (CDRs)) that connect the beta chains with one another and are exposed to the solvent. There are at least three of these cycles on each bank of the beta-sheet sandwich, where the bank is the limit of the protein perpendicular to the direction of the beta chains (see US Pat. No. 6,818,418). These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, comprising the entire unit of antigen recognition in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics the antigen binding properties that are of a similar nature and affinity to those of the antibodies. These scaffolds can be used in a strategy of random selection and in vitro cycle mixing, which is similar to the affinity maturation process of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds wherein the cycle regions of the molecule can be replaced with the complementarity determining regions (CDRs) of the invention using conventional cloning techniques.

The technology of ankyrin is based on the use of proteins with repetitive modules derived from ankyrin as scaffolding to support the variable regions, which can be used to link to different objectives. The ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel helices and one b-turn. The linkage of the variable regions is optimized for the most part using the ribosome display.

Avimeres are derived from the protein that contains the natural A domain, such as HER3. These domains are used by nature for protein-protein interactions, and in humans, more than 250 proteins are structurally based on A-domains. Avimers consist of a number of different "A-domain" monomers (2-10) linked via amino acid linkers. Avimeres that can be linked to a target antigen can be created using the methodology described, for example, in the United States of America Patent Application Publication Numbers 20040175756; 20050053973; 20050048512; and 20060008844.

Affibody affinity ligands are small single proteins composed of a three-helix bundle based on the scaffolding of one of the IgG binding domains of protein A. Protein A is a surface protein made from the bacterium Staphylococcus aureus. This scaffolding domain consists of 58 amino acids, 13 of which are randomly selected to generate Affibody libraries with a large number of ligand variants (see, for example, U.S. Patent No. US 5,831, 012). The Affibody molecules mimic the antibodies, they have a molecular weight of 6 kDa, compared to the molecular weight of the antibodies, which is 150 kDa. Despite its small size, the binding site of Affibody molecules is similar to that of an antibody.

The anticalinas are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widely spread group of small, robust proteins that are usually involved in the transport or physiological storage of chemically sensitive or insoluble compounds. Several natural lipocalins are present in the tissues or in the human body fluids. The architecture of the protein reminds the immunoglobulins, with hypervariable cycles on top of a rigid structure. However, in contrast to antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acid residues, which is only marginally greater than a single immunoglobulin domain. The set of four cycles, which forms the linking buchaca, shows a pronounced structural plasticity and tolerates a variety of side chains. The binding site, therefore, can be reconfigured into a registered process for the purpose of recognizing the target molecules prescribed differently with high affinity and specificity. A protein of the lipocalin family, the biline binding protein (BBP) of Pieris Brassicae, has been used to develop anticalines by mutagenizing the set of four cycles.

An example of a patent application describing anticalines is in International Publication of TCP Number WO 199916873.

Affilin molecules are small proteins that are not immunoglobulin, which are designed to have specific affinities for proteins and small molecules. The new afilin molecules can be selected very quickly from two libraries, each of which is based on a different scaffolding protein derived from the human being. Affilin molecules do not show any structural homology with immunoglobulin proteins. Currently, two afilin scaffolds, one of which is gamma-crystalline, a human structural protein of the eye lens, and the other is that of the proteins of the "ubiquitin" superfamily. Both human scaffolds are very small, show a high stability to temperature, and are almost resistant to changes in pH and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of the gamma-crystalline-derived proteins are described in International Publication Number W0200104144, and examples of the "ubiquitin-like" proteins are described in International Publication Number WO 2004106368.

Protein epitope mimics (PEMs) are cyclic peptide molecules of medium size (MW 1 -2kDa) that mimic secondary structures of beta-hairpin proteins, the main secondary structure involved in protein-protein interactions .

In some embodiments, the Fabs are converted to the silent I g G 1 format by changing the Fe region. For example, the antibodies in Table 1 can be converted to the IgG format.

Human or humanized antibodies The present invention provides completely human antibodies that specifically bind to a HER3 protein (eg, human HER3 and / or cynomolgus / mouse / rat).

Compared with chimeric or humanized antibodies, human HER3 antibodies or fragments thereof also have reduced antigenicity when administered to human subjects.

Human HER3 antibodies or fragments thereof can be generated using methods that are known in the art. For example, the "humaneering" (human design) used to convert non-human antibodies into human designed antibodies. The Patent Publication of the United States of America Number 20050008625 describes an in vivo method for replacing a non-human antibody variable region with a human variable region in an antibody, while maintaining the same binding characteristics or providing better binding characteristics in relation to those of the non-human antibody. The method relies on the epitope-guided replacement of the variable regions of a non-human reference antibody with a fully human antibody. The resulting human antibody is generally structurally unrelated to the reference non-human antibody, but binds to the same epitope on the same antigen as the reference antibody. Briefly stated, the approach of complementary replacement guided by the serial epitope is made possible by establishing a competition in the cells between a "competitor" and a library of various hybrid antibodies of the reference ("test antibodies") to bind to limiting amounts of antigen in the presence of a reporter system that responds to the binding of the test antibody to the antigen. The competitor can be the reference antibody or a derivative thereof, such as a single chain Fv fragment. The competitor may also be a natural or artificial ligand of the antigen that binds to the same epitope as the reference antibody. The competitor's only requirements are that it binds to the same epitope as the reference antibody, and that it competes with the reference antibody for binding to the antigen. The test antibodies have a V-region of antigen binding in common from the non-human reference antibody, and the other V-region is randomly selected from a diverse source, such as a repertoire library of the human antibodies. . The common V-region from the reference antibody serves as a guide, placing the test antibodies on the same epitope on the antigen, and in the same orientation, such that selection is forced towards the highest binding fidelity. to the antigen with the reference antibody.

Many types of reporter systems can be used to detect the desired interactions between the test antibodies and the antigen. For example, reporter fragments complementary to the antigen and the test antibody can be linked, respectively, in such a way that only the activation of the reporter is present through the complementation of the fragments when the test antibody is linked to the antigen. When the antibody-test-antigen-reporter fragment fusions are co-expressed with a competitor, the activation of the reporter becomes dependent on the ability of the test antibody to compete with the competitor, which is proportional to the affinity of the test antibody by the antigen. Other reporter systems that may be used include the reactivator of a self-inhibited reporter reactivation system (RAIR), as disclosed in United States of America Patent Application Serial Number 10 / 208,730 (Publication Number 20030198971), or the competitive activation system disclosed in United States of America Patent Application Serial Number 10 / 076,845 (Publication Number 20030157579).

With the complementarity replacement system guided by the serial epitope, the selection is made to identify the cells that express a single test antibody together with the competitor, antigen, and reporter components. In these cells, each test antibody competes one-to-one with the competitor to bind to a limiting amount of antigen. The activity of the reporter is proportional to the amount of antigen bound to the test antibody, which in turn is proportional to the affinity of the test antibody for the antigen and the stability of the test antibody. The test antibodies are initially selected based on their activity in relation to that of the reference antibody when expressed as the test antibody. The result of the first round of selection is a set of "hybrid" antibodies, each of which is comprised of the same non-human V-region from the reference antibody and a human V-region from the library, and each of which binds to the same epitope on the antigen as the reference antibody. One or more of the hybrid antibodies selected in the first round will have an affinity for the antigen comparable with, or greater than, that of the reference antibody.

In the second step of V-region replacement, the human V-regions selected in the first step are used as a guide for the selection of human replacements for the V-region of the remaining non-human reference antibody with a diverse library of human V regions cognate. The hybrid antibodies selected in the first round can also be used as competitors for the second round of selection. The result of the second round of selection is a set of fully human antibodies that differ structurally from the reference antibody, but which compete with the reference antibody to bind to the same antigen. Some of the selected human antibodies bind to the same epitope on the same antigen as the reference antibody. Among these selected human antibodies, one or more is linked to the same epitope with an affinity that is comparable to, or greater than, that of the reference antibody.

Using one of the mouse or chimeric HER3 antibodies, or fragments thereof, described above as the reference antibody, this method can be easily employed to generate human antibodies that bind to human HER3 with the same binding specificity and the same or better link affinity. In addition, these human HER3 antibodies or fragments thereof can also be obtained commercially in companies that customarily produce human antibodies, for example, in KaloBios, Inc. (Mountain View, CA).

CAMELID ANTIBODIES The antibody proteins obtained from the members of the camel and dromedary family (Camelus bactrianus and Camelus dromedarius), including members of the new world, such as llama species (Lama paceos, Lama glama and lama vicugna), they have been characterized with respect to size, structural complexity, and antigenicity for human subjects. Certain IgG antibodies from this family of mammals, as found in nature, lack light chains and, therefore, are structurally distinct from the typical four-chain quaternary structure that has two heavy chains and two light chains, for the antibodies of other animals. See International Publication of the TCP Number PCT / EP93 / 02214 (International Publication Number WO 94/04678 published on March 3, 1994).

A region of the camelid antibody, which is the only small variable domain identified as VH H, can be obtained by genetic engineering to provide a small protein that has a high affinity for a target, which results in a protein derived from low antibody. molecular weight known as a "camelid nanobody". See U.S. Patent No. 5,759,808 issued June 2, 1998; see also Stijlemans et al. (2004) J Biol Chem 279: 1256-1261; Dumoulin et al (2003) Nature 424: 783-788; Pleschberger et al. (2003) Bioconjugate Chem 14: 440-448; Cortez-Retamozo et al. (2002) Int J Cancer 89: 456-62; and Lauwercys et al. (1998) EMBO J 17: 3512-3520. Designed libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium (for example, US Pat. Nos. US200601 15470, Domantis US20070065440, US20090148434). As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered in a recombinant manner to obtain a sequence that closely resembles a human sequence, ie, the nanobody can be "humanized". Accordingly, the low natural antigenicity of camelid antibodies for humans can be further reduced.

The camelid nanobody has a molecular weight of about one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to the larger antibody proteins, ie, the camelid nanobodies are useful as reagents for detecting antigens that are otherwise cryptically, using the classical immunological techniques, and as possible therapeutic agents. Accordingly, yet another consequence of the small size is that a camelid nanobody can be an inhibitor as a result of binding to a specific site in a narrow slot or slit of a target protein and, therefore, can serve in a capacity that resembles closer to the function of a classic low molecular weight drug, than that of a classical antibody.

The low molecular weight and compact size also result in camelid nanobodies that are extremely thermostable, stable at extreme pHs and proteolytic digestion, and poorly antigenic. Another consequence is that the camelid nanobodies move easily from the circulatory system to the tissues, and even cross the blood-brain barrier and can treat the disorders that affect the nervous tissue. Nanobodies can further facilitate the transport of drugs through the blood-brain barrier. See U.S. Patent Application Number 20040161738 published August 19, 2004. These characteristics, combined with the low antigenicity for humans, indicate the great therapeutic potential. In addition, these molecules can be fully expressed in prokaryotic cells, such as from E. coli, and are expressed as fusion proteins with bacteriophages, and are functional.

In accordance with the foregoing, a feature of the present invention is an antibody or camelid nanobody that has a high affinity for HER3. In certain embodiments herein, the camelid antibody or nanobody is naturally produced in the camelid animal, that is, it is produced by the camelid following immunization with HER3 or with a peptide fragment thereof, using the techniques described in FIG. present for other antibodies. Alternatively, the camelid nanobody that binds to HER3 is designed, that is, produced, by a selection, for example, from a phage library that exhibits the appropriately mutagenized camelid nanobody proteins, using methods of panning with LRP6 as a target, as described in the Examples herein. The designed nanobodies can also be made custom-made by genetic engineering to have a half-life in a receiving subject of 45 minutes to two weeks. In a specific modality, the antibody or nanobody of camelid is obtained by grafting the sequences of the complementarity determining regions (CDRs) of the heavy or light chain of the human antibodies of the invention, into the nanobody or single-domain antibody structure sequences, as described , for example, in the International Publication of TCP Number PCT / EP93 / 02214. In one embodiment, the camelid antibody or nanobody is linked to at least the amino acid residue in domain 2 of H ER3 selected from amino acids 265-277 and 315. In one embodiment, the camelid antibody or nanobody is linked at least the amino acid residue Lys 268 in domain 2 of HER3.

Bispecific molecules and multivalent antibodies In another aspect, the present invention provides biparatopic, bispecific or multispecific molecules comprising an antibody or a fragment thereof that binds to an epitope within domain 2 of HER3. The antibody or a fragment thereof can be derived or linked to another functional molecule, for example, to another peptide or protein (eg, to another antibody or ligand to a receptor), to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody or a fragment thereof can, in fact, be derived or linked to more than one other functional molecule to generate biparatopic or multispecific molecules that bind to more than two different binding sites and / or target molecules; such biparatópicas or multispecific molecules. For creating a bispecific molecule, an antibody or a fragment thereof can be functionally linked (eg, by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule results.

Other clinical benefits can be provided by linking two or more antigens within an antibody (Coloma et al. (1997); Merchant et al. (1998); Alt et al. (1999); Zuo et al. (2000); Lu et al. (2004), Lu et al. (2005), Marvin et al. (2005), Marvin et al. (2006), Shen et al. (2007), Wu et al. (2007), Dimasi et al. (2009), Michaelson et al. 2009)). (Morrison et al. (1997) Nature Biotech 15: 159-163; Alt et al. (1999) FEBS Letters 454: 90-94; Zuo et al. (2000) Protein Engineering 13: 361-367; Lu et al. (2004) JBC 279: 2856-2865; Lu et al. (2005) JBC 280: 19665-19672; Marvin et al. (2005) Acta Pharmacologica Sinica 26: 649-658; Marvin et al. (2006) Curr Opin Drug Disc Develop 9: 184-193; Shen et al. (2007) J Immun Methods 218: 65-74; Wu et al. (2007) Nat Biotechnol. 1 1: 1290-1297; Dimasi et al. (2009) J Mol Biol. 393: 672-692; and Michaelson et al. (2009) mAbs 1: 128-141.

Bispecific molecules can be prepared by conjugation of constitutive binding specificities, using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately, and then conjugated with each other, for example, a variety of coupling or crosslinking agents can be used for covalent conjugation. Examples of the crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl thioacetate (SATA), 5,5'-dithiobis- (2-nitro-benzoic acid) (DTNB), o-phenylene -dimaleimide (oPDM), N-succinimidyl-3- (2-pyridyldithio) -propionate (SPDP), and sulfosuccinimidyl-4- (N-maleimido-methyl) -cyclohexan-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu et al. (1985) Proc. Nati. Acad. Sci. USA 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78: 1 18-132; Brennan et al. (1985) Science 229: 81-83), and Glennie et al. (1987) J. Im munol. 139: 2367-2375). The conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

With the antibodies, they can be conjugated by sulfhydryl linkage of the articulation regions of the C-terminus of the two heavy chains. In a particular embodiment, the articulation region is modified to contain a non-sulfhydryl residue number, eg, one, before conjugation.

In an alternative way, both binding specificities can be encoded in the same vector, and can be expressed and assemble in the same host cell. This method is particularly useful when the bispecific molecule is a fusion protein of mAb x mAb, mAb x Fab, Fab x F (ab ') 2 or ligand x Fab. A bispecific molecule of the invention can be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules can comprise at least two molecules of a single chain. Methods for the preparation of bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881, 175; 5, 132, 405; 5,091, 513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

The binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (eg, growth inhibition), or Western blotting. Blot. Each of these assays broadly detects the presence of the protein-antibody complexes of a particular interest, by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

In another aspect, the present invention provides multivalent compounds comprising at least two identical or different fragments of the antibodies that bind to HER3.

The antibody fragments can be linked together by means of protein fusion or by means of a covalent or non-covalent bond. The tetravalent compounds can be obtained, for example, by cross-linking the antibodies of the invention with an antibody that binds to the constant regions of the antibodies of the invention, for example, Fe or the region of articulation. The trimerizing domain is described, for example, in Borean, European Patent Number EP 1012280B 1. Pentamerizing modules are described, for example, in International Publication of TCP Number PCT / EP97 / 05897.

In one embodiment, a biparatopic / bispecific antibody binds to the amino acid residues within domain 2 of HER3.

In another embodiment, the invention pertains to dual-function antibodies, wherein a single monoclonal antibody has been modified in such a way that the antigen binding site binds to more than one antigen, such as a dual-function antibody that binds both HER3 and another antigen (for example, HER 1, HER2, and HER4). In another embodiment, the invention pertains to a dual-function antibody that targets antigens having the same conformation, for example, an antigen having the same conformation of HER3 in the "closed" or "inactive" state. Examples of antigens with the same conformation of HER3 in the "closed" or "inactive" state include, but are not limited to, HER1 and HER4. Accordingly, a double-function antibody can bind to HER3 and HER1; to HER3 and HER4, or to HER 1 and HER4. The Double-bond specificity of the double-function antibody can be further translated into a double activity, or in activity inhibition. (See, for example, Jenny Bostrom et al. (2009) Science: 323; 1610-1614).

Antibodies with a long half-life The present invention provides antibodies that specifically bind to an epitope within domain 2 of HER3, which have a prolonged half-life.

Many factors can affect the half-life of a protein in vivo. Examples are renal filtration, metabolism in the liver, degradation by proteolytic enzymes (proteases), and immunogenic responses (eg, neutralization of the protein by antibodies and uptake by macrophages and dendritic cells) . A variety of strategies can be employed to extend the half-life of the antibodies of the present invention. For example, by chemical bonding to polyethylene glycol (PEG), reCODE PEG, antibody scaffolding, poly-sialic acid (PSA), hydroxyethyl starch (HES), albumin binding ligands, and carbohydrate protectants; By genetic fusion to proteins that bind to serum proteins, such as albumin, IgG, FcRn, and transferrin; by coupling (genetic or chemical) with other binding fractions that bind to serum proteins, such as nanobodies, Fabs, DARPins, avimers, Affibodies, and anticalines; by genetic fusion with rPEG, albumin, domain of albumin, protein binding to albumin, and Fe; or by incorporation in nanovehicles, slow release formulations, or medical devices.

In order to prolong the serum circulation of the antibodies in vivo, the inert polymeric molecules, such as the high molecular weight PEG, can be bound to the antibodies or to a fragment thereof, with or without a multifunctional linker, and either through the specific conjugation of the PEG site to the N or C terminus of the antibodies, or through the epsilon-amino groups present on the lysine residues. For pegylation, an antibody, the antibody, or the fragment thereof, is typically reacted with polyethylene glycol (PEG), such as a reactive ester derivative or PEG aldehyde, under conditions wherein one or more PEG groups become bound to the antibody or fragment of the antibody. PEGylation can be carried out by an acylation reaction or by an alkylation reaction with a reactive PEG molecule (or an analogous reactive water soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the PEG forms that have been used to derive other proteins, such as mono alkoxy (1-10 carbon atoms) - or aryloxy-polyethylene glycol or polyethylene glycol- maleimide In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. The derivation of the linear or branched polymer that results in a minimal loss of biological activity will be used. The degree of conjugation can be monitored tightly by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecules with the antibodies. Unreacted PEG can be separated from the PEG-antibody conjugates by size exclusion or ion exchange chromatography. PEG-derived antibodies can be tested for their binding activity, as well as their efficacy in vivo, using methods well known to those skilled in the art, for example, by the immunoassays described herein. Methods for pegylating proteins are known in the art, and can be applied to the antibodies of the invention. See, for example, European Patent Number EP 0 154 316 by Nishimura et al., And European Patent Number EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include the orthogonal chemically engineered engineering reconstitution technology (ReCODE PEG), which incorporates chemically specified side chains into the biosynthetic proteins, by means of a reconstituted system that includes the tRNA synthetase and the tRNA. This technology makes possible the incorporation of more than 30 new amino acids in the biosynthetic proteins in E.coli, yeast, and mammalian cells. The tRNA incorporates a non-active amino acid in any place where an amber codon is placed, converting the amber from a stop codon to one that signals the incorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) is also You can use it to prolong the serum half-life. This technology involves genetically fusing an unstructured protein tail of 300 to 600 amino acids with an existing pharmaceutical protein. Because the apparent molecular weight of this unstructured protein chain is approximately 15 times greater than its actual molecular weight, the serum half-life of the protein greatly increases. In contrast to traditional PEGylation, which requires chemical conjugation and re-purification, the manufacturing process is greatly simplified, and the product is homogeneous.

Poly-sialylation is another technology, which uses the natural poly-sialic acid (PSA) polymer to prolong the active life and improve the stability of peptides and therapeutic proteins. Poly-sialic acid (PSA) is a polymer of sialic acid (a sugar). When used for drug delivery of therapeutic proteins and peptides, poly-sialic acid provides a protective micro-environment in the conjugation. This increases the active life of the therapeutic protein in the circulation, and prevents it from being recognized by the immune system. The poly-sialic acid polymer (PSA) is found naturally in the human body. It was adopted by certain bacteria that evolved over millions of years to coat their walls with it. Then these naturally poly-sialylated bacteria were able, by virtue of molecular imitation, to coat the body's defense system. The poly-sialic acid (PSA), the latest in protective technology of the nature, can be easily produced from these bacteria in large quantities and with previously determined physical characteristics. Bacterial sialic poly-acid (PSA) is completely non-immunogenic, even when coupled to proteins, because it is chemically identical to poly-sialic acid (PSA) of the human body.

Another technology includes the use of starch derivatives of h id roxi-eti lo ("HES") linked to the antibodies. Hydroxyethyl starch (HES) is a modified natural polymer derived from waxy corn starch, and can be metabolized by the body's enzymes. Hydroxyethyl starch (HES) solutions are usually administered to replace the deficient blood volume and to improve the Theological properties of the blood. The HESylation of an antibody makes it possible to prolong the half-life in the circulation, by increasing the stability of the molecule, as well as by reducing the renal elimination, resulting in an increase in biological activity. By varying different parameters, such as the molecular weight of hydroxyethyl starch (HES), a wide range of hydroxyethyl starch (HES) antibody conjugates can be custom-made.

Antibodies that have an increase in their half-life can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into a constant domain of IgG, or a FcRn binding fragment thereof (preferably a Fe or Fe domain fragment). See, for example, International Publication Number WO 98/23289; International Publication Number WO 97/34631; and U.S. Patent Number 6,277,375.

In addition, the antibodies can be conjugated with albumin in order to make the antibody or the antibody fragment more stable in vivo or have a longer half-life in vivo. Techniques are well known in the art, see, for example, International Publications Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent Number EP 413,622.

The HER3 antibody or a fragment thereof can also be fused with one or more of human serum albumin (HSA) polypeptides, or a portion thereof. Human serum albumin (HSA), a protein of 585 amino acids in its mature form, is responsible for a significant proportion of the serum osmotic pressure, and also functions as a vehicle for endogenous and exogenous ligands. The function of albumin as a carrier molecule and its inert nature are desirable properties to be used as a vehicle and polypeptide transporter in vivo. The use of albumin as the component of an albumin fusion protein as a vehicle for various proteins has been suggested in International Publication Number WO 93/15199, in International Publication Number WO 93/15200, and in European Patent Number EP 413 622. It has also been proposed to use the N-terminal fragments of human serum albumin (HSA) for fusions with polypeptides (European Patent Number EP 399 666). In accordance with the above, by means of the fusion or genetic or chemical conjugation of the antibodies or fragments thereof with albumin, the half-life can be stabilized or extended, and / or the activity of the molecule can be preserved for periods of time prolonged in solution, in vitro and / or in vivo.

Fusion of the albumin with another protein can be achieved by genetic manipulation, such that the DNA encoding human serum albumin (HSA), or a fragment thereof, binds to the DNA encoding the protein. A suitable host is then transformed or transfected with the fused nucleotide sequences, arranged in such a manner on a suitable plasmid, that they express a fusion polypeptide. Expression can be effected in vitro, for example, from prokaryotic or eukaryotic cells, or in vivo, for example, from a transgenic organism. Additional methods pertaining to fusions of human serum albumin (HSA) can be found, for example, in International Publications Nos. WO 2001077137 and WO 200306007, incorporated herein by reference. In a specific embodiment, the expression of the fusion protein is carried out in mammalian cell lines, for example, in CHO cell lines. The altered differential binding of an antibody to a receptor at low or high pHs is also contemplate within the scope of the invention. For example, the affinity of an antibody can be modified such that it remains bound to its receptor at a low pH, for example, low pH within a lysosome, by modifying the antibody to include additional amino acids, such as a histidine. in a complementarity determining region (CDR) of the antibody (see, for example, Tomoyuki Igawa et al. (2010) Nature Biotechnology; 28, 1203-1207).

Antibody conjugates The present invention provides antibodies or fragments thereof that specifically bind to recombinantly fused or chemically conjugated HER3 (including both covalent and non-covalent conjugates) to a heterologous protein or polypeptide (or fragment thereof), preferably to a polypeptide of at least 10, of at least 20, of at least 30, of at least 40, of at least 50, of at least 60, of at least 70, of at least 80, of at least 90 or at least 100 amino acids) to generate fusion proteins. In particular, the invention provides fusion proteins comprising an antibody fragment described herein (eg, a Fab fragment, an Fd fragment, an Fv fragment, an F (ab) 2 fragment, a VH domain, a VH CDR , a VL domain or a VL CDR), and a heterologous protein, polypeptide, or peptide. Methods for fusing or conjugating proteins, polypeptides, or peptides to an antibody or a fragment of antibodies are known in the art. See, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patents Nos. EP 307,434 and EP 367, 166; International Publications Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al. (1991) Proc. Nati Acad. Sci. USA 88: 10535-10539; Zheng et al. (1995) J. Immunol. 154: 5590-5600; and Vil et al. (1992) Proc. Nati Acad. Sci. USA 89: 1 1337-1 1341.

Additional fusion proteins can be generated through the techniques of gene mixing, mixture of motifs, mixture of exons, and / or mixture of codons (collectively referred to as "DNA mixture"). The DNA mixture can be used to alter the activities of the antibodies of the invention or fragments thereof (for example, antibodies or fragments thereof with higher affinities and lower dissociation rates). See, in general terms, U.S. Patent Nos. 5,605,793; 5.81 1, 238; 5,830,721; 5,834,252; and 5,837,458; Patten et al. (1997) Curr. Opinion Biotechnol. 8: 724-33; Harayama, (1998) Trends Biotechnol. 16 (2): 76-82; Hansson et al. (1999) J. Mol. B i or 1. 287: 265-76; and Lorenzo and Blasco, (1998) Biotechniques 24 (2): 308-313 (each of these patents and publications is incorporated herein by reference in its entirety). The antibodies or fragments thereof, or the encoded antibodies or fragments thereof, they can be altered by subjecting them to random mutagenesis by polymerase chain reaction (PCR) susceptible to error, random insertion of nucleotide, or by other methods before recombination. A polynucleotide that encodes an antibody or a fragment thereof that specifically binds to a HER3 protein can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies or fragments thereof can be fused with marker sequences, such as a peptide, in order to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the label provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1), among others, many of which are commercially available. As described in Gentz et al. (1989) Proc. Nati Acad. Sci. USA 86: 821-824, for example, hexa-histidine provides convenient purification of the fusion protein. Other brands of peptides useful for purification include, but are not limited to, the hemagglutinin label ("HA"), which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) Cell. 37: 767), and the "flag" mark.

In other embodiments, the antibodies of the present invention or fragments thereof, are conjugated with a diagnostic or detectable agent. These antibodies can be useful for monitor or forecast the establishment, development, progress and / or severity of a disease or disorder as part of a clinical trial procedure, such as determining the effectiveness of a particular therapy. This diagnosis and detection can be carried out by coupling the antibody with detectable substances, including, but not limited to, various enzymes, such as, but not limited to, red radicle peroxidase, alkaline phosphatase, beta-galactosidase, or acetyl -cholinesterase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichloro-triazinyl-amine-fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; luminescent biomaterials, such as, but not limited to, luciferase, luciferin, and aquorine; radioactive materials, such as, but not limited to, iodine (131 l, 125 l, 123l, and 1211,), carbon (14C), sulfur (35S), tritium (3H), indium (115ln, 113l n, 112I n, and 11 11 n,), teenecio (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum ("Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd , 149Pm, 140La, 175Yb, 166hlo, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97RU, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, and 117Tin, and metals Positron emitters using different positron emission tomographies, and non-radioactive paramagnetic metal ions.

The present invention also encompasses the uses of the antibodies or fragments thereof conjugated with a therapeutic moiety. An antibody or fragment thereof can be conjugated to a therapeutic moiety, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g. alpha-emitters. A cytotoxin or a cytotoxic agent includes any agent that is detrimental to the cells.

In addition, an antibody or fragment thereof can be conjugated to a therapeutic moiety or a drug moiety that is modified to give a biological response. Therapeutic fractions or drug fractions should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein, peptide, or polypeptide possessing a desired biological activity. These proteins may include, for example, a toxin, such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein, such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, an apoptotic agent, an anti-angiogenic agent; or, a biological response modifier, such as, for example, a lymphokine. In one embodiment, the HER3 antibody or a fragment thereof, is conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. These conjugates are referred to herein as "Immunoconjugates". The immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins". A cytotoxin or a cytotoxic agent includes any agent that is detrimental to (eg, kills) the cells. Examples include taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicine, doxorubicin, daunorubicin, dihydroxy-anthrazine-dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercapto-purine, 6-thioguanine, cytarabine, 5-fluoro-uracil-decarbazine), ablating agents (e.g., mechlorethamine, thiotepa-chlorambucil, melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromo-mannitol, streptozotocin, mitomycin C, and cis-dichloro-diamine-platinum (II) (DDP) cisplatin, anthracyclines (eg, daunorubicin (formerly daunomycin), and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, vincristine and vinblastine). (See, for example, Seattle Genetics, Patent of the United States of America Number US20090304721).

Other examples of therapeutic cytotoxins that can be conjugated with an antibody of the invention or with a fragment of the same, include duocarmycins, calicheamicins, maytansins and auristatins, and derivatives thereof. An example of a conjugated calicheamicin antibody is commercially available (Mylotarg ™, Wyeth-Ayerst).

The cytotoxins can be conjugated with the antibodies of the invention or with the fragments thereof, using the linkology technology available in the art. Examples of the types of linkers that have been used to conjugate a cytotoxin to an antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides and linkers that contain peptides. A linker can be selected, i.e., for example, that is susceptible to dissociation by a low pH within the lysosomal compartment, or that is susceptible to dissociation by proteases, such as proteases preferentially expressed in tumor tissue, such such as cathepsins (for example, cathepsins B, C, D).

For an additional discussion of the types of cytotoxins, linkers and methods for conjugating the therapeutic agents to the antibodies, see also Saito et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail et al. (2003) Cancer Immunol. Immunother. 52: 328-337; Payne, (2003) Cancer Cell 3: 207-212; Alien, (2002) Nat. Rev. Cancer 2: 750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53: 247-264.

The antibodies of the present invention or the fragments of they can also be conjugated with a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radiommunoconjugates. Examples of radioactive isotopes that can be conjugated with antibodies for diagnostic or therapeutic use include, but are not limited to, iodine131, indium1 1 1, yttrium90, and lutetium177. The methods for the preparation of radioimmunoconjugates are established in the matter. Examples of radioimmunoconjugates are commercially available, including Zevalin ™ (DEC Pharmaceuticals), and Bexxar ™ (Corixa Pharmaceuticals), and similar methods can be employed to prepare radioimmunoconjugates using the antibodies of the invention. In certain embodiments, the macrocyclic chelate is 1 acid, 4,7,10-tetra-azaciclododecan-N, N ', N ", N'" - tetra-acetic acid (DOTA) which can be attached to the antibody through of a linker molecule. These linker molecules are commonly known in the art and are described in Denardo et al. (1998) Clin Cancer Res. 4 (10): 2483-90; Peterson et al. (1999) Bioconjug. Chem. 10 (4): 553-7; and Zimmerman et al. (1999) Nucí. Med. Biol. 26 (8): 943-50, each incorporated as reference in its entirety.

The téenicas for conjugating therapeutic moieties to antibodies are well known, see, eg, Arnon et al, "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al (Editors), pages 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies for Drug Delivery", in Controlled Drug Delivery (2nd Edition), Robinson et al. (Editors), pages 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies 84: Biological and Clinical Applications, Pinchera et al. (Editors), pages 475-506 (1985); "Analysis, Results, and Future Prospective of The Therapeutic Use of Radiolabeled Antibody in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al. (Editors), pages 303-16 (Academic Press 1985), and Thorpe et al. (1982) Immunol. Rev. 62: 1 19-58.

The antibodies can also be attached to solid supports, which are particularly useful for immunoassays or for the purification of the target antigen. These solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Antibody combinations In another aspect, the invention pertains to HER3 antibodies, or fragments thereof of the invention, used with other therapeutic agents, such as other antibodies, small molecule inhibitors, mTOR inhibitors, or PI3 kinase inhibitors. Examples include, but are not limited to, the following: HER1 inhibitors: HER3 antibodies or fragments thereof can be used with HER1 inhibitors, which include, but are not limited to, Matuzumab (EMD72000), Erbitux® / Cetuximab (ImClone), Vectibix® / panitumumab (Amgen), mAb 806, and Nimotuzumab (TheraCIM), lressa® / Gefitinib (Astrazeneca); CI-1033 (PD183805) (Pfizer), Lapatinib (GW-572016) (GlaxoSmithKline), Tykerb / lapatinib ditosylate (SmithKline Beecham), Tarceva / Erlotinib HCL (OSI-774) (OSI Pharmaceuticals), and PKI-166 (Novartis), and N- [4 - [(3-chloro-4-fluoro-phenyl) -amino] -7 - [[(3"S") - tetrahydro-3-furanyl] -oxy] -6-qumazolinyl ] -4- (dimethylamino) -2-butenamide, sold under the tradename Tovok® by Boehringer Ingelheim).

HER2 Inhibitors: HER3 antibodies or fragments thereof, can be used with HER2 inhibitors, which include, but are not limited to, Pertuzumab (sold under the registered trademark Omnitarg® by Genentech), Trastuzumab ( sold under the registered trademark Herceptin® by Genentech / Roche), MM-1 1 1, neratinib (also known as HKI-272, (2 E) - N- [4 - [[3-chloro-4 - [(pyrid i h-2-i I) -methoxy] -phenyl] -amino] -3-cyano-7-ethoxy-quinolin-6-yl] -4- (dimethylamino) -but-2-enamide, and described in International Publication of TCP Number WO 05/028443), lapatinib or Lapatinib Ditosylate (sold under the registered trademark Tykerb® by GlaxoSmithKline.

HER3 Inhibitors: HER3 antibodies or fragments thereof can be used with HER3 inhibitors, which include, but are not limited to, MM-121, MM-11 1, IB4C3, 2DID12 (U3 Pharma AG) , AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech), and small molecules that inhibit HER3.

HER4 Inhibitors: HER3 antibodies or fragments thereof, can be used with HER4 inhibitors.

PI3K Inhibitors: HER3 antibodies or fragments thereof, can be used with PI3 kinase inhibitors, which include, but are not limited to, 4- [2- (1 H-indazol-4-yl) - 6 - [[4- (Methyl-sulfonyl) -piperazin-1-yl] -methyl] -thieno- [3,2-d] -pyrimidin-4-yl] -morpholine (also known as GDC 0941 and described in International Publications of TCP Numbers WO 09/036082 and WO 09/055730), 2-methyl-2- [4- [3-methyl-2-oxo-8- (qumolin-3-yl) -2,3-dihydro-imidazo- [4,5 -c] -quinol-n-1-yl] -phenyl] -propionitrile (also known as BEZ 235 or NVP-BEZ 235, and described in International Publication of the TCP Number WO 06/122806), BMK120 and BYL719.

MTOR Inhibitors: HER3 antibodies or fragments thereof, can be used with mTOR inhibitors, which include, but are not limited to, Temsirolimus (sold under the trade name Torisel® by Pfizer), ridaforolimus (formally known as deferolimus, dimethyl-phosphinate of (1 R, 2R, 4S) -4 - [(2R) -2 - [(1 R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R) -1, 18-dihydroxy-19,30-dimethoxy-15,17,21, 23,29, 35-hexamethyl-2,3,10,14,20-pentaoxo-1 1, 36-dioxa-4-aza-tricyclo- [30.3.1.04, 9] -hexatriaconta- 16, 24,26, 28-tetraen-12-yl] -propyl] -2-methoxy-cyclohexyl, also known as Deferolimus , AP23573 and M K8669 (Ariad Pharm.), And described in International Publication of TCP Number WO 03/064383), everolimus (RAD001) (sold under the tradename Afinitor® by Novartis). One or more therapeutic agents can be administered either simultaneously or before or after the administration of a HER3 antibody or a fragment thereof of the present invention.

Methods for the production of the antibodies of the invention (i) Nucleic acids encoding the antibodies The invention provides substantially purified nucleic acid molecules that encode polypeptides comprising segments or domains of the HER3 antibody chains described above. Some of the nucleic acids of the invention comprise the nucleotide sequence encoding the heavy chain variable region of the HER3 antibody and / or the nucleotide sequence encoding the light chain variable region. In a specific embodiment, the nucleic acid molecules are those identified in Table 1. Some other nucleic acid molecules of the invention comprise nucleotide sequences that are substantially identical (eg, at least 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent) to the nucleotide sequences of those identified in Table 1. When expressed from the appropriate expression vectors, the polypeptides encoded for these polynucleotides are capable of exhibiting binding capacity to the HER3 antigen.

Also provided in the invention are polynucleotides that encode at least one complementarity determining region (CDR) and usually all three complementarity determining regions (CDRs) from the heavy or light chain of the antibody or fragment thereof stipulated above. Some other polynucleotides encode all or substantially all of the variable region sequence of the heavy chain and / or light chain of the antibody or fragment thereof stipulated above. Due to the degeneracy of the code, a variety of nucleic acid sequences will encode each of the amino acid sequences of inm unoglobulin.

The nucleic acid molecules of the invention can encode both a variable region and a constant region of the antibody. Some of the nucleic acid sequences of the invention comprise nucleotides that encode a mature heavy chain variable region sequence that is substantially identical (eg, at least 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent) to the mature heavy chain variable region sequence of an HER3 antibody, stipulated in Table 1. Some other nucleic acid sequences comprising nucleotides that encode a sequence of the mature light chain variable region that is substantially identical (eg, at least 80 percent, 90 percent, 95 percent, 96 percent, 97 percent, 98 percent, or 99 percent) to the sequence of the region mature light chain variable of a HER3 antibody stipulated in Table 1.

The polynucleotide sequences can be produced by de novo solid phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of an existing sequence encoding the antibody the fragment thereof. The direct chemical synthesis of nucleic acids can be carried out by methods known in the art, such as the phospho-triester method of Narang et al. (1979) Meth. Enzymol. 68: 90; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109; the diethyl phosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859; and the solid support method of U.S. Patent Number 4,458,066. The introduction of mutations to a polynucleotide sequence by polymerase chain reaction (PCR) can be carried out as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Editor), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Editors), Academic Press, San Diego, CA, 1990; Mattila et al. (1991) Nucleic Acids Res. 19: 967; and Eckert et al. (1991) PCR Methods and Applications 1: 17.

Expression vectors and host cells are also provided in the invention to produce the antibodies or fragments thereof. Different expression vectors can be used to express the polynucleotides encoding the antibody chains or fragments thereof. Both viral and non-viral-based expression vectors can be used in order to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette to express a protein or RNA, and human artificial chromosomes (see, for example, Harrington et al. (1997) Nat Genet 15: 345). For example, non-viral vectors useful for the expression of polynucleotides and polypeptides that bind to HER3 in mammalian (eg, human) cells include pTioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C, (Invitrogen, San Diego, CA), M PSV vectors, and numerous other vectors known in the art to express other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, Herpes viruses, SV40-based vectors, papilloma virus, HBP Epstein Barr virus, vaccine virus vectors, and Semliki Forest virus (SFV). See, Brent et al. (1995) supra \ Smith, Annu. Rev. Microbiol. 49: 807; and Rosenfeld et al. (1992) Cell 68: 143.

The choice of expression vector depends on the intended host cells in which the vector is to be expressed.

Typically, expression vectors contain a promoter and other regulatory (e.g., enhancer) sequences that are operably linked to the polynucleotides that encode a chain of the antibody or a fragment thereof. In some embodiments, an inducible promoter is employed to prevent expression of the inserted sequences, except under induction conditions. Inducible promoters include, for example, arabinose, lacZ, the metallothionein promoter, or a heat shock promoter. The cultures of the transformed organisms can be expanded under non-inducing conditions without forcing the population to encode sequences whose expression products are better tolerated by the host cells. In addition to the promoters, other regulatory elements may also be required or desired for the efficient expression of an antibody chain or a fragment thereof. These elements typically include an ATG start codon and the adjacent ribosome binding site, or other sequences. In addition, the efficiency of expression can be improved by the inclusion of appropriate enhancers for the cellular system in use (see, for example, Scharf et al. (1994) Results Probl Cell Differ 20: 125 and Bittner et al. 1987) Meth. Enzymol., 153: 516). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

Expression vectors can also provide a secretory signal sequence position to form a fusion protein with the polypeptides encoded by the inserted sequences of the antibody or fragment. Plus frequently, the inserted sequences of the antibody or fragment are linked to the signal sequences before being included in the vector. Vectors to be used for the purpose of receiving the sequences encoding the heavy and light chain variable domains of the antibody or fragment sometimes also encode the constant regions or parts thereof. These vectors allow the expression of the variable regions as fusion proteins, leading the constant regions in this way to the production of the intact antibodies or fragments thereof. Typically, these constant regions are human.

The host cells for receiving and expressing the chains of the antibody or fragment can be either prokaryotic or eukaryotic. E. coli is a prokaryotic host useful for the cloning and expression of the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and different species of Pseudomonas. In these prokaryotic hosts, expression vectors can also be made, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from lambda phage. Promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, to initiate and perform transcription and translation. Other microbes, such as yeast, can also be used to express the antibodies or fragments thereof. Insect cells can also be used in combination with baculovirus vectors.

In some preferred embodiments, mammalian host cells are used to express and produce the antibodies or fragments thereof. For example, can be any of a hybridoma cell line expressing the endogenous immunoglobulin genes, or a mammalian cell line harboring an exogenous expression vector. These include any normal or normal or abnormal immortal mortal or animal cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins, including CHO cell lines, different Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells, and hybridomas have been developed. The use of mammalian tissue cell culture to express polypeptides is discussed in general terms, for example, in Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y. , N .Y. , 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, eg, Queen et al. (1986) Immunol. 89: 49-68), and the sites of necessary processing information, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, specific to the cell type, stage specific, and / or modulable or adjustable. Useful promoters include, but are not limited to, the metallothionein promoter, the major late promoter constitutive adenovirus, the promoter of MMTV inducidle dexamethasone, the SV40 promoter, the MRP pollll, the constitutive MPSV promoter, the CMV promoter induced by tetracycline (such as the immediate-early human CMV promoter), the constitutive promoter of CMV, and the promoter-enhancer combinations known in the art.

Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, transfection with calcium chloride is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electroporation for other cellular hosts can be used. (See generally Sambrook et al., Supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and micromjection, ballistic methods, virosomes, inmunoMposomas conjugated polycation: nucleic acid, naked DNA, artificial virions, fusion structural VP22 protein of Herpes virus (Elliot and O'Hare (1997) Cell 88: 223), DNA uptake potentiated by the agent, and ex vivo transduction. For the long-term high yield production of recombinant proteins, stable expression will often be desired. For example, cell lines stably expressing the chains of antibodies or fragments can be prepared, using the expression vectors of the invention containing viral replication origins or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, the cells can be allowed to grow for 1 to 2 days in an enriched media before switching to the selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the sequences introduced in the selective medium. Stably transfected resistant cells can be proliferated using tissue culture techniques appropriate for the cell type. (ii) Generation of the monoclonal antibodies of the invention Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example, the conventional Kohler somatic cell hybridization technique and Milstein, (1975) Nature 256: 495. Many techniques can be used to produce monoclonal antibodies, for example, viral or oncogenic transformation of B-lymphocytes.

An animal system for the preparation of hybridomas is the murine system. The production of hybridoma in the mouse is a well-established procedure. Immunization protocols and techniques for the isolation of splenocytes immunized for fusion are known in the art. Fusion components (eg, murine myeloma cells) and fusion methods are also known.

The chimeric or humanized antibodies of the present invention can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. The DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest, and is designed to contain non-murine (e.g., human) immunoglobulin sequences, using conventional molecular biology techniques. For example, to create a chimeric antibody, murine variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.) . To create a humanized antibody, the murine complementarity determining (CDR) regions can be inserted into a human structure using known methods in the technical. See, for example, U.S. Patent No. 5225539 to Winter, and U.S. Patent Nos. 5530101; 5585089; 5693762 and 6180370 to Queen et al.

In a certain embodiment, the antibodies of the invention are human monoclonal antibodies. These human monoclonal antibodies directed against HER3 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system in place of the mouse system. These transgenic and transchromosomal mice include the mice referred to herein as HuMAb mice and KM mice, respectively. , and are collectively referred to herein as "human Ig mice".

The HuMAb® mouse (Medarex, Inc.) contains miniloci of the human immunoglobulin gene encoding human immunoglobulin sequences not reconfigured heavy chain (myg) and light K, together with targeted mutations that inactivate the endogenous myk chain loci (see, for example, Lonberg et al. (1994) Nature 368 (6474): 856-859). In accordance with the foregoing, the mice exhibit a reduced expression of mouse IgM ok, and in response to immunization, the introduced heavy and light chain human transgenes undergo class change and somatic mutation to generate high human IgGx monoclonal antibodies. affinity (Lonberg et al. (1994) supra revised in Lonberg, (1994) Handbook of Experimental Pharmacology 1 13: 49-101; Lonberg and Huszar, (1995) Intern. Rev. Immunol. 13: 65-93, and Harding and Lonberg, (1995) Ann. N. Y. Acad. Sci. 764: 536-546). The preparation and use of HuMAb mice, and the genomic modifications carried by these mice, is further described in Taylor et al. (1992) Nucleic Acids Research 20: 6287-6295; Chen et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Nati Acad. Sci. USA 94: 3720-3724; Choi et al. (1993) Nature Genetics 4: 1 17-123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 2912-2920; Taylor et al. (1994) International Immunology 579-591; and Fishwild et al. (1996) Nature Biotechnology 14: 845-851, the content of all of which is specifically incorporated herein by reference in its entirety. See also, Patents of the United States of North America Nos. 5,545,806; 5,569,825; 5,625, 126; 5,633,425; 5,789,650; 5,877,397; 5,661, 016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent Number 5,545,807 to Surani et al .; International Publications of TCP Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 971 13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and the International Publication of TCP Number WO 01/14424 to Korman et al.

In another embodiment, the human antibodies of the invention can be reproduced using a mouse carrying sequences of human immunoglobulin on transgenes and transchromosomes, such as a mouse carrying the human heavy chain transgene and a human light chain transchromosome. These mice, referred to herein as "KM mice", are described in detail in International Publication of the TCP Number WO 02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art, and can be used to reproduce the antibodies that bind to the HER3 of the invention. For example, an alternative transgenic system referred to as the Xeno-mouse (Abgenix, Inc.) may be used. These mice are described, for example, in U.S. Patent Nos. 5,939,598; 6,075, 181; 6,1, 14,598; 6, 150,584 and 6, 162,963 to Kucherlapati et al.

Moreover, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to reproduce the antibodies that bind to the HER3 of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice" can be used.; these mice are described in Tomizuka et al. (2000) Proc. Nati Acad. Sci. USA 97: 722-727. In addition, carriers of heavy and light chain transchromosomes have been described in the technique. human (Kuroiwa et al. (2002) Nature Biotechnology 20: 889-894), and can be used to reproduce the HER3 antibodies of the invention.

The human monoclonal antibodies of the invention can also be prepared using phage display methods to screen libraries of human immunoglobulin genes. These phage display methods for isolating human antibodies are established in the art or are described in the Examples below. See, for example: Patents of the United States of North America Nos. 5,223,409; 5,403,484; and 5,571, 698 to Ladner et al; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al .; Patents of the United States of North America Nos. 5,969, 108 and 6, 172, 197 to McCafferty et al .; and Patents of the United States of North America Nos. 5,885,793; 6,521, 404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

The human monoclonal antibodies of the invention can also be prepared using SCID mice in which human immune cells have been reconstituted, which human immune cells have been reconstituted in such a way that a human antibody response can be generated after immunization. These mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al. (iii) Design of Structure or Faith The designed antibodies of the invention include those wherein modifications have been made to the structure residues within the VH and / or the VL, for example, to improve the properties of the antibody. Typically these structural modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "retromue" one or more structure residues up to the corresponding germline sequence. More specifically, an antibody that has been subjected to a somatic mutation may contain structure residues that differ from the germline sequence from which the antibody is derived. These residues can be identified by comparing the structure sequences of the antibody with the germline sequences from which the antibody is derived. In order to return to the sequences of the structure region up to their germline configuration, somatic mutations can be "back-mutated" to the germline sequence, for example, by site-directed mutagenesis. It is intended that these "retromutated" antibodies also be encompassed by the invention.

Another type of structure modification involves mutating one or more residues within the framework region, or even within one or more complementarity determining regions (CDR), to remove the T-cell epitopes in order to reduce in this manner the potential immunogenicity of the antibody. East This approach is also referred to as "deimmunization" and is described in greater detail in U.S. Patent Publication Number 20030153043 by Carr et al.

In addition or in an alternative manner to the modifications made within the framework regions (FR) or the complementarity determining regions (CDR), the antibodies of the invention can be designed to include modifications within the Fe region, typically for altering one or more functional properties of the antibody, such as serum half-life, complement fixation, binding to the Fe receptor, and / or antigen-dependent cellular cytotoxicity. Additionally, an antibody of the invention can be chemically modified (e.g., one or more chemical moieties can be bound to the antibody), or it can be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these modalities is described in more detail below. The numbering of waste in the Fe region is that of the Kabat European Union index.

In one embodiment, the joint region of CH 1 is modified such that the number of cysteine residues in the joint region is altered, for example, that it increases or decreases. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al; the number of cysteine residues in the region of CH 1 joint is altered, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fe-articulation region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fc-joint fragment, such that the antibody has a damaged link to Staphylococcal protein A (SpA) in relation to the SpA binding of the Fc-articulation native domain. This approach is described in greater detail in U.S. Patent Number 6, 165,745 by Ward et al.

In yet other embodiments, the Fe region is altered, by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids may be replaced with a different amino acid residue, such that the antibody has an altered affinity for an effector ligand, but retains the ability to bind the antigen of the parent antibody. The effector ligand with which the affinity is altered can be, for example, a Fe receptor or the C 1 complement component. This approach is described in greater detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from the amino acid residues, can be replaced with a different amino acid residue, such that the antibody has an altered linkage of C 1 and / or a complement-dependent cytotoxicity (CDC). ) reduced or abolished. This approach is described in greater detail in U.S. Patent Number 6, 194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to bind the complement. This approach is further described in International Publication of TCP Number WO 94/29351 by Bodmer et al.

In still another embodiment, the Fe region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcy receptor, by modifying one or more amino acids . This approach is further described in International Publication of TCP Number WO 00/42072 by Presta. Moreover, the binding sites on human I g G 1 have been mapped for FcyRI, FcyRIl, FcyRIII and FcRn, and variants with a better binding have been described (see Shields et al. (2001) J. Biol. Chen. 276: 6591-6604).

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (ie, the antibody lacks glycosylation). The Glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. These carbohydrate modifications can be carried out, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more glycosylation sites from the variable region structure to thereby eliminate glycosylation at that site. This aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in greater detail in the Patents of the United States of North America Nos. 5,714,350 and 6,350,861 by Co and collaborators Additionally or alternatively, an antibody having an altered type of glycosylation, such as a hypophosphorylated antibody having reduced amounts of fucosyl residues, or an antibody having an increase in the GIcNac bisection structures can be made. It has been shown that these altered glycosylation patterns increase the ability of antibody-dependent cellular cytotoxicity (ADCC) of the antibodies. These carbohydrate modifications can be carried out, for example, by expressing the antibody in a host cell with an altered glycosylation mask. Cells with an altered glycosylation machinery have been described in this field, and can be used as host cells wherein the recombinant antibodies of the invention are expressed, for thus producing an antibody with altered glycosylation. For example, European Patent Number EP 1, 176, 195 by Hang et al. Describes a cell line with a functionally altered FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in this cell line exhibit hypophosphorylation. International Publication of TCP Number WO 03/035835 by Presta describe a variant CHO cell line, Lecl3 cells, with a reduced ability to bind fucose to carbohydrates linked with Asn (297), also resulting in hypophospylation of the antibodies expressed in that host cell (see also Shields et al. (2002) J. B io I. Chem. 277: 26733-26740). International Publication of TCP Number WO 99/54342 by Umana et al. Describes cell lines designed to express glycoprotein-modifying glycosyl transferases (eg, beta (1, 4) -N acetyl-glucosaminyl-transferase III (G n TI 11)), in such a way that the antibodies expressed in the designed cell lines exhibit an increase in the GIcNac bisection structures, which results in an increase in the antibody dependent cellular cytotoxicity (ADCC) activity of the antibodies (see also Umana et al. (1999) Nat. Biotech 17: 176-180).

In another embodiment, the antibody is modified to increase its biological half-life. Different approaches are possible. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in the Patent of the United States of America Number 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CH 1 or CL region to contain a salvage receptor binding epitope taken from two cycles of a CH 2 domain of a Fe region. an IgG, as described in the Patents of the United States of North America Nos. 5,869,046 and 6, 121, 022 by Presta et al. (iv) Methods to design altered antibodies The HER3 antibodies or fragments thereof of the invention, having the VH and VL sequences or the full length heavy and light chain sequences shown herein, can be used to create new HER3 antibodies by modifying the heavy chain and / or full length light chain sequences, the VH and / or VL sequences, or the constant regions attached thereto. Accordingly, in another aspect of the invention, the structural features of a HER3 antibody or fragment thereof are used to create structurally related HER3 antibodies that retain at least a functional property of the antibodies of the invention, such as the link to human HER3, and also the inhibition of one or more functional properties of HER3. For example, one or more complementarity determining regions (CDR) of the antibodies of the present invention, or mutations thereof, can be combine in a recombinant manner with the known structure regions and / or with other complementarity determining regions (CDRs), to create additional recombinantly designed HER3 antibodies, as discussed above. Other types of modifications include those described in the previous section. The starting material for the design method is one or more of the VH and / or VL sequences provided herein, or one or more complementarity determining regions (CDRs) thereof. To create the designed antibody, it is not necessary to actually prepare (ie, express as a protein) an antibody having one or more of the VH and / or VL sequences provided herein, or one or more complementarity determining regions (CDR) ) from the same. Instead, the information contained in the sequences is used as the starting material to create a "second generation" sequences derived from the original sequences, and then the "second generation" sequences are prepared, and they are expressed as a protein.

In accordance with the foregoing, in another embodiment, the invention provides a method for the preparation of an antibody consisting of: a heavy chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 2, 22, 42, 62, 82, 102, 122, 142, 162, 182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, and 522; a sequence of CDR2 selected from the group consisting of SEQ ID NOs: 3, 23, 43, 63, 83, 103, 123, 143, 163, 183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, and 523; and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 4, 24, 44, 64, 84, 104, 124, 144, 164, 184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, and 524; and a light chain variable region antibody sequence having a CDR1 sequence selected from the group consisting of SEQ ID NOs: 8, 28, 48, 68, 88, 108, 128, 148, 168, 188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, and 528; a sequence of CDR2 selected from the group consisting of SEQ ID NOs: 9, 29, 49, 69, 89, 109, 129, 149, 169, 189, 209, 229, 249, 269, 289, 309, 329 , 349, 369, 389, 409, 429, 449, 469, 489, 509, and 529; and / or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 70, 90, 1 10, 130, 150, 170, 190, 210, 230, 250, 270, 290 , 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, and 530; altering at least one amino acid residue within the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to create at least one altered antibody sequence; and expressing the altered antibody sequence as a protein. The altered antibody sequence can also be prepared by screening libraries of antibodies having the fixed CDR3 sequences or minimum essential binding determinants as described in U.S. Patent No. US2005 / 0255552, and diversity in the CDR1 and CDR2 sequences. The screening can be carried out in accordance with any suitable screening technology for screening antibodies from antibody libraries, such as phage display technology.

Conventional molecular biology techniques can be employed to prepare and express the altered antibody sequence. The antibody encoded by the altered antibody sequence is one that retains one, some, or all of the functional properties of the antibodies or fragments thereof described herein, whose functional properties include, but are not limited to, specifically the binding to the antibody. HER3 human and / or cynomolgus; the antibody binds to HER3 and inhibits the biological activity of HER3 by inhibiting HER signaling activity in a phospho-H ER assay.

The functional properties of the altered antibodies can be evaluated using conventional assays available in the art and / or described herein, such as those stipulated in the Examples (eg, ELISAs).

In certain embodiments of the methods of designing the antibodies of the invention, mutations may be introduced in a random or selective manner, together with all or part of a coding sequence of an antibody or fragment, and the HER3 antibodies may be screened. resulting modifications for determine their binding activity and / or other functional properties, as described herein. Mutation methods have been described in this field. For example, International Publication of TCP Number WO 02/092780 by Short discloses methods for creating and screening antibody mutations using saturation metatagenesis, synthetic linkage assembly, or a combination thereof. Alternatively, International Publication of TCP Number WO 03/074679 by Lazar et al., Describes methods for using computer tracking methods to optimize the physico-chemical properties of antibodies. Characterization of the antibodies of the invention The antibodies of the invention can be characterized by different functional assays. For example, they can be characterized by their ability to inhibit biological activity by inhibiting HER signaling in a phospho-HER assay, as described herein, by their affinity to a HER3 protein (e.g., HER3). human and / or cynomolgus), by the link to the epitope, by its resistance to proteolysis, and by its ability to block signaling downstream of HER3. Different methods can be used to measure signaling mediated by HER3. For example, the signaling pathway of HER3 can be monitored by: (i) the measurement of phospho-HER3; (ii) measuring the phosphorylation of HER3 or other downstream signaling proteins (eg, Akt); (iii) ligand blocking assays, as described herein; (iv) training of heterodimer; (v) genetic expression signature dependent on HER3; (vi) internalization of the receiver; and (vii) cellular phenotypes driven by HER3 (eg, proliferation).

The ability of an antibody to bind to HER3 can be detected by labeling the antibody of interest directly, or the antibody can be unmarked and the linkage detected indirectly using different sandwich assay formats known in the art.

In some embodiments, the HER3 antibodies block or compete with the binding of a reference HER3 antibody, with a HER3. These may be the fully human HER3 antibodies described above. They may also be other mouse chimeric, or humanized, HER3 antibodies that bind to the same epitope as the reference antibody. The ability to block or compete with the binding of the reference antibody indicates that an HER3 antibody under test binds to the same epitope or to an epitope similar to that defined by the reference antibody, or to an epitope that is sufficiently close to the bound epitope by the reference HER3 antibody. These antibodies have special probabilities of sharing the convenient properties identified for the reference antibody. The ability to block or compete with the reference antibody can be determined, for example, by a competition binding assay. With a competition binding assay, the antibody under test is examined for determining its ability to inhibit the specific binding of the reference antibody to a common antigen, such as a polypeptide or a HER3 protein. A test antibody competes with the reference antibody for the specific binding to the antigen if an excess of the test antibody substantially inhibits the binding of the reference antibody. Substantial inhibition means that the test antibody reduces the specific binding of the reference antibody usually by at least 10 percent, 25 percent, 50 percent, 75 percent, or 90 percent.

There are a number of known competition binding assays that can be used to evaluate the competition of a HER3 antibody, with the reference HER3 antibody, to bind to a HER3. These include, for example, direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al. (1983) Methods in Enzymology 9: 242-253 ); direct enzyme immunoassay (EIA) of solid phase biotin-avidin (see Kirkland et al. (1986) J. Immunol., 137: 3614-3619); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow and Lane, supra); radioimmunoassay (RIA) direct labeling in solid phase using the 1-125 mark (see Morel et al. (1988) Molec. Immunol. 25: 7-15); direct enzyme immunoassay (EIA) of biotin-avidin in solid phase (Cheung et al. (1990) Virology 176: 546-552); and direct-labeled radiommunoassay (RIA) (Moldenhauer et al. (1990) Scand, J. Immunol., 32: 77-82). Typically, this assay involves the use of the purified antigen bound to a solid surface or to cells carrying any of them, an antibody that binds to the unlabeled test HER3, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or to the cells in the presence of the test antibody. Usually, the test antibody is present in excess. Antibodies identified by the competition assay (competing antibodies), include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antibody so that the steric hindrance is present.

In order to determine whether selected H ER3 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (e.g., reagents from Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be carried out using ELISA plates coated with the HER3 polypeptide. The binding of biotinylated monoclonal antibodies (mAbs) it can be detected with a streptavidin-alkaline phosphatase probe. To determine the isotype of an antibody that binds to purified HER3, isotype ELISAs can be carried out. For example, the wells of the microtitre plates can be coated with 1 microgram / milliliter of anti-human IgG overnight at 4 ° C. After blocking with 1 percent bovine serum albumin (BSA), the plates are reacted with 1 microgram / milliliter or less of the HER3 monoclonal antibody or purified isotype controls, at room temperature, for one to two hours . The wells can then be reacted with either human IgG 1 or the conjugated probes with alkaline phosphatase specific for human IgM. The plates are then developed and analyzed in such a manner that the isotype of the purified antibody can be determined.

In order to demonstrate the binding of HER3 monoclonal antibodies to living cells expressing a H ER3 polypeptide, flow cytometry can be used. Briefly stated, cell lines expressing HER3 (grown under conventional growth conditions) can be mixed with different concentrations of an antibody that binds to HER3 in phosphate-buffered serum (PBS) containing bovine serum albumin ( BSA) at 0.1 percent and fetal calf serum at 10 percent, and incubated at 4 ° C for 1 hour. After washing, the cells are reacted with the fluorescein-labeled anti-human IgG antibody under the same conditions as the dyeing of the primary antibody. Samples can be analyzed by the FACScan instrument using the light and side scattering properties to give gate to the individual cells. An alternative assay can be employed using fluorescence microscopy (in addition to, or in place of) the flow cytometry assay. The cells can be stained exactly as described above, and can be examined by fluorescence microscopy. This method allows the visualization of individual cells, but can reduce sensitivity, depending on the density of the antigen.

The antibodies of the invention or fragments thereof can be further tested for their reactivity with a HER3 polypeptide or an antigenic fragment by Western blot. Briefly stated, purified polypeptides or HER3 fusion proteins, or cell extracts can be prepared from cells expressing HER3, and subjected to sodium dodecyl sulfate / polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 10 percent fetal calf serum, and probed with the monoclonal antibodies to be tested. The binding of human IgG can be detected using anti-human IgG / alkaline phosphatase, and revealed with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

A number of readings can be used to evaluate the efficacy and specificity of HER3 antibodies in cell-based assays of heterodimer formation induced by the ligand. The activity can be evaluated by one or more of the following: (i) Inhibition of heterodimerization of HER2 induced by the ligand, with other members of the EGF family in a target cell line, eg, MCF-7 breast cancer cells. The immunoprecipitation of the HER2 complexes from the cellular ones can be carried out with an antibody specific for the receptor, and the absence / presence of other EGF receptors and their biologically relevant ligands can be analyzed within the complex, following electrophoresis / Western blotting, by probing with antibodies to other EGF receptors. (ii) Inhibition of the activation of the signaling pathways by the heterodimers activated by the ligand. The association with HER3 seems to be a key for other members of the EGF receptor family, to elicit the maximum cellular response following ligand binding. In the case of the defective kinase HER3, HER2 provides a functional tyrosine kinase domain to make it possible for signaling to occur following the binding of the growth factor ligands. Accordingly, cells that co-express HER2 and HER3 can be treated with the ligand, eg, heregulin, in the absence and in the presence of the inhibitor, and the effect on tyrosine phosphorylation of HER3 is monitored by a number of ways , including immunoprecipitation of H ER3 from the treated treated cells and the subsequent Western blot using anti-phosphotyrosine antibodies (see Agus, op cit for details). Alternatively, a high production assay can be developed by trapping HER3 from the solubilized ones on the wells of a 96-well plate coated with an anti-HER3 receptor antibody, and the level of phosphorylation of HER3 is measured. tyrosine using, for example, anti-phosphotyrosine antibodies labeled with europium, as incorporated by Waddleton et al. (2002) Anal. Biochem. 309: 150-157.

In a broader extension of this approach, effector molecules that are known to be activated downstream of activated receptor heterodimers, such as mitogen-activated protein kinases (MAPK) and Akt, can be directly analyzed by immunoprecipitation from of the used treated, and making the transfer with the antibodies that detect the activated forms of these proteins, or by analyzing the ability of these proteins to modify / activate the specific substrates. (iii) Inhibition of cell proliferation induced by the ligand. It is known that a variety of cell lines co-express combinations of ErbB receptors, for example, many breast and prostate cancer cell lines. The assays can be carried out in formats of 24/48/96 wells, with the reading based around DNA synthesis (incorporation of tritiated thymidine), increase in the number of cells (crystal violet staining), etc.

A number of readings can be used to evaluate the efficacy and specificity of HER3 antibodies in cell-based assays of ligand-independent homo- and hetero-dimer formation. For example, over-expression of HER2 triggers activation of the ligand-independent kinase domain as a result of spontaneous dimer formation. The over-expressed HER2 generates either homo- or hetero-dimers with other HER molecules, such as HER1, HER3 and HER4.

The ability of the antibodies or fragments thereof to block in vivo growth of tumor xenografts from human tumor cell lines, of which their tumorigenic phenotype is known to be at least partially dependent on the activation of the tumor signaling. heterodimer of HER3 through the ligand, for example, BxPC3 pancreatic cancer cells, etc. This can be evaluated in immunocompromised mice, either alone or in combination with a cytotoxic agent appropriate for the cell line in question. Examples of functional tests are also described in the Examples section below.

Prophylactic and therapeutic uses The present invention provides methods for the treatment of a disease or a disorder associated with the HER3 signaling pathway by administering to a subject in need thereof, an effective amount of the antibody of the invention or of a fragment of it. In a specific embodiment, the present invention provides a method for the treatment or prevention of cancers (e.g., breast cancer, colo-rectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral tumors of the nerve sheath, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophageal cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, hyperplasia benign prostatic (BPH), gynecomastia and endometriosis), by administering to a subject in need thereof, an effective amount of the antibodies of the invention or fragments thereof. In some embodiments, the present invention provides methods for the treatment or prevention of cancers associated with a HER3 signaling pathway, by administering to a subject in need thereof, an effective amount of the antibodies of the invention.

In a specific embodiment, the present invention provides methods for the treatment of cancers associated with a HER3 signaling pathway including, but not limited to, breast cancer, colorectal cancer, lung cancer, multiple myeloma, cancer of ovary, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, leukemia chronic myeloid, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophageal cancer, glioblastoma, soft tissue clear cell sarcoma, mesothelioma malignant, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prosthetic hyperplasia (BPH), gynecomastia, and endometriosis.

The antibodies or fragments thereof of the invention can also be used to treat or prevent other disorders associated with aberrant or defective HER3 signaling, including, but not limited to, respiratory diseases, osteoporosis, osteoarthritis, polycystic kidney disease, diabetes , schizophrenia, vascular disease, heart disease, non-oncogenic proliferative diseases, fibrosis, and neurodegenerative diseases, such as Alzheimer's disease.

Suitable agents for the combination treatment with HER3 antibodies include conventional care agents known in the art that are capable of modulating the ErbB signaling pathway. Suitable examples of conventional care agents for HER2 include, but are not limited to, Herceptin and Tykerb. Suitable examples of conventional care agents for EG FR include, but are not limited to, Iressa, Tarceva, Erbitux and Vectibix, as described above. Other agents that may be suitable for the Combination treatment with HER3 antibodies include, but are not limited to, those that modulate receptor tyrosine kinases, G-protein coupled receptors, the pathway of growth / survival signal transduction, nuclear hormone receptors, apoptotic pathways, the cell cycle, and angiogenesis.

Diagnostic Uses In one aspect, the invention encompasses diagnostic assays for determining the expression of the HER3 protein and / or nucleic acid, as well as the function of the HER3 protein, in the context of a biological sample (e.g., blood, serum, cells, tissue), or from an individual suffering from cancer, or at risk of developing cancer.

Diagnostic assays, such as competitive assays, rely on the ability of a labeled analogue (the "tracer") to compete with the analyte in the test sample for the limited number of binding sites in a common link component. . The linkage component is generally insolubilized before or after the competition, and then the tracer and the analyte linked to the linkage component are separated from the unlinked tracer and analyte. This separation is carried out by decanting (where the binding component was previously insolubilized) or by centrifugation (wherein the binding component was precipitated after the competitive reaction). The amount of analyte in the test sample is inversely proportional to the amount of linked tracer, as measured by the amount of marker substance. The dose response curves are prepared with known amounts of the analyte, and compared with the test results, in order to quantitatively determine the amount of analyte present in the test sample. These assays are referred to as ELISA systems when enzymes are used as the detectable markers. In an assay in this manner, competitive binding between antibodies and HER3 antibodies results in the bound HER3, preferably the HER3 epitopes of the invention, which is a measure of the antibodies in the serum sample, more particularly, of the inhibition of the antibodies in the serum sample.

A significant advantage of the assay is that the inhibition measurement of the antibodies is made directly (ie, those interfering with the HER3 binding, in a specific manner, the epitopes). This assay, particularly in the form of an ELISA, has considerable applications in the clinical environment and in routine blood screening.

Another aspect of the invention provides methods for determining the expression of the HER3 nucleic acid or the activity of HER3 in an individual, to thereby select the appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allow the selection of agents (for example, drugs) for the therapeutic or prophylactic treatment of an individual based on the genotype of the individual (for example, the genotype of the individual examined to determine the individual's ability to respond to a particular agent).

Yet another aspect of the invention pertains to monitoring the influence of agents (eg, drugs) on the expression or activity of HER3 in clinical studies. Pharmaceutical Compositions To prepare the sterile or pharmaceutical compositions including the antibodies or fragments thereof, these antibodies or fragments thereof are mixed with a pharmaceutically acceptable carrier or excipient. The compositions may additionally contain one or more other therapeutic agents that are suitable for the treatment or prevention of cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, schwannial peripheral nerve sheath tumors, head and neck cancer, bladder cancer, esophageal cancer, Barrett's esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal and melanoma cancer, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis).

The formulations of therapeutic and diagnostic agents are they can be prepared by mixing with vehicles, excipients, or physiologically acceptable stabilizers in the form, for example, of freeze-dried powders, aqueous pastes, aqueous solutions, lotions, or suspensions (see, for example, Hardman et al. (2001) Goodman and Gilman , The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis and collaborators (Editors) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al.

(Editors) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (Editors) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

The selection of a regimen of administration for a therapeutic product depends on several factors, including the rate of rotation of the serum or tissue of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, a regimen of administration maximizes the amount of therapeutic product delivered to the patient in a manner consistent with an acceptable level of side effects. In accordance with the above, the quantity of biological product supplied depends in part on the particular entity and the severity of the condition that is trying. The guide is available to select the appropriate doses of antibodies, cytokines, and small molecules (see, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, United Kingdom; Kresina (Editor) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y .; Bach (Editor) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003) New Engl. J. Med. 348: 601-608; Milgrom et al. (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344: 783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342: 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602).

The determination of the appropriate dose is made by the clinician, for example, using parameters or factors that are known or suspected in the matter that affect the treatment or that are predicted to affect the treatment. Generally speaking, the dose begins with a quantity a little less than the optimal dose, and is increased by small increments thereafter until the desired or optimal effect is achieved in relation to any negative side effects. Important diagnostic measures include those of the symptoms, for example, of inflammation or the level of inflammatory cytokines produced.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied such as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toto the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the index of excretion of the particular compound that is used, the duration of the treatment, other drugs, the compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and of similar factors known in medical technology.

The compositions comprising the antibodies or fragments thereof of the invention can be provided by continuous infusion, or by doses at intervals, for example, one day, one week, or 1 to 7 times per week. The dose may be given intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. A specific dosage protocol is one that involves the maximum dose or a dosage frequency that avoids significant undesirable side effects. A total weekly dose it can be at least 0.05 micrograms / kilogram of body weight, from at least 0.2 micrograms / kilogram, of at least 0.5 micrograms / kilogram, of at least 1 microgram / kilogram, of at least 10 micrograms / kilogram, of at least 100 micrograms / kilogram, of at least 0.2 milligrams / kilogram, of at least 1.0 milligram / kilogram, of at least 2.0 milligrams / kilogram, of at least 10 milligrams / kilogram, of at least 25 milligrams / kilogram, or of at least 50 milligrams / kilogram (see, for example, Yang et al. (2003) New Engl. J. Med. 349: 427-434; Herold et al. (2002) New Engl. J. Med. 346: 1692-1698; Liu et al. (1999) ) J Neurol Neurosurg, Psych 67: 451-456, Portielji et al. (2003) Cancer Immunol Immunother 52: 133-144). The desired dose of the antibodies or fragments thereof is approximately the same as for an antibody or polypeptide, on a basis of moles / kilogram of body weight. The desired plasma concentration of the antibodies or fragments thereof is approximately the same as for an antibody, on a basis of moles / kilogram of body weight. The dose may be at least 15 micrograms, from at least 20 micrograms, from at least 25 micrograms, from at least 30 micrograms, from at least 35 micrograms, from at least 40 micrograms, from at least 45 micrograms, from at least 50 micrograms, of at least 55 micrograms, of at least 60 micrograms, at least 65 micrograms, of at least 70 micrograms, of at least 75 micrograms, of at least 80 micrograms, of at least 85 micrograms, of at least 90 micrograms, of at least 95 micrograms, or of at least 100 micrograms. The doses administered to a subject can amount to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more.

For the antibodies or fragments thereof of the invention, the dosage administered to a patient can be from 0.0001 milligrams / kilogram to 100 milligrams / kilogram of the patient's body weight. The dosage can be between 0.0001 milligrams / kilogram and 20 milligrams / kilogram, between 0.0001 milligrams / kilogram and 10 milligrams / kilogram, between 0.0001 milligrams / kilogram and 5 milligrams / kilogram, between 0.0001 and 2 milligrams / kilogram, of between 0.0001 and 1 milligram / kilogram, between 0.0001 milligrams / kilogram and 0.75 milligrams / kilogram, between 0.0001 milligrams / kilogram and 0.5 milligrams / kilogram, between 0.0001 milligrams / kilogram and 0.25 milligrams / kilogram, between 0.0001 and 0.15 milligrams / kilogram, between 0.0001 and 0.10 milligrams / kilogram, between 0.001 and 0.5 milligrams / kilogram, between 0.01 and 0.25 milligrams / kilogram, or between 0.01 and 0.10 milligrams / kilogram of the patient's body weight.

The dosage of the antibodies or fragments thereof of the invention can be calculated using the weight of the patient in kilograms (kg) multiplied by the doses to be administered in milligrams / kilogram. The dosage of the antibodies or fragments thereof of the invention, may be 150 micrograms / kilogram or less, 125 micrograms / kilogram or less, 100 micrograms / kilogram or less, 95 micrograms / kilogram or less, 90 micrograms / kilogram or less, of 85 micrograms / kilogram or less, of 80 micrograms / kilogram or less, of 75 micrograms / kilogram or less, of 70 micrograms / kilogram or less, of 65 micrograms / kilogram or less, of 60 micrograms / kilogram or less, of 55 micrograms / kilogram or less, 50 micrograms / kilogram or less, 45 micrograms / kilogram or less, 40 micrograms / kilogram or less, 35 micrograms / kilogram or less, 30 micrograms / kilogram or less, 25 micrograms / kilogram or less, 20 micrograms / kilogram or less, 15 micrograms / kilogram or less, 10 micrograms / kilogram or less, 5 micrograms / kilogram or less, 2.5 micrograms / kilogram or less, 2 micrograms / kilogram or less, 1.5 micrograms / kilogram or less, 1 microgram mo / kilogram or less, 0.5 micrograms / kilogram or less, or 0.5 micrograms / kilogram or less, of the patient's body weight.

The unit dose of the antibodies or fragments thereof of the invention can be from 0.1 milligrams to 20 milligrams, from 0.1 milligrams to 15 milligrams, from 0.1 milligrams to 12 milligrams, from 0.1 milligrams to 10 milligrams, from 0.1 milligrams to 8 milligrams. milligrams, from 0.1 milligrams to 7 milligrams, from 0.1 milligrams to 5 milligrams, from 0.1 to 2.5 milligrams, from 0.25 milligrams to 20 milligrams milligrams, from 0.25 to 15 milligrams, from 0.25 to 12 milligrams, from 0.25 to 10 milligrams, from 0.25 to 8 milligrams, from 0.25 milligrams to 7 milligrams, from 0.25 milligrams to 5 milligrams, from 0.5 milligrams to 2. 5 milligrams, from 1 milligram to 20 milligrams, from 1 milligram to 15 milligrams, from 1 milligram to 12 milligrams, from 1 milligram to 10 milligrams, from 1 milligram to 8 milligrams, from 1 milligram to 7 milligrams, from 1 milligram to 5 milligrams milligrams, or from 1 milligram to 2.5 milligrams.

The dosage of the antibodies or fragments thereof of the invention can reach a serum titration of at least 0.1 micrograms / milliliter, of at least 0.5 micrograms / milliliter, of at least 1 microgram / milliliter, of at least 2 micrograms / milliliter, of at least 5 micrograms / milliliter, of at least 6 micrograms / milliliter, of at least 10 micrograms / milliliter, of at least 15 micrograms / milliliter, of at least 20 micrograms / milliliter, of at least 25 micrograms / milliliter, of at least 50 micrograms / milliliter, of at least 100 micrograms / milliliter, of at least 125 micrograms / milliliter, of at least 150 micrograms / milliliter, of at least 175 micrograms / milliliter, of at least 200 micrograms / milliliter, of at least 225 micrograms / milliliter, of at least 250 micrograms / milliliter, of at least 275 micrograms / milliliter, at least 300 micrograms / milliliter, of at least 325 micrograms / milliliter, of at least 350 micrograms / milliliter, of at least 375 micrograms / milliliter, or at least 400 micrograms / milliliter, in a subject. Alternatively, the dosage of the antibodies or fragments thereof of the invention, can reach a serum titration of at least 0.1 micrograms / milliliter, of at least 0.5 micrograms / milliliter, of at least 1 microgram / milliliter, of at least 2 micrograms / milliliter, of at least 5 micrograms / ml, at least 6 micrograms / milliliter, of at least 10 micrograms / milliliter, of at least 15 micrograms / milliliter, of at least 20 micrograms / milliliter, of at least 25 micrograms / milliliter, of at least 50 micrograms / milliliter, of at least 100 micrograms / milliliter, of at least 125 micrograms / milliliter, of at least 150 micrograms / milliliter, of at least 175 micrograms / milliliter, of at least 200 micrograms / milliliter, of at least 225 micrograms / milliliter, of at least 250 micrograms / milliliter, of at least 275 micrograms / milliliter, of at least 300 micrograms / milliliter, of which at least 325 micrograms / milliliter, of at least 350 micrograms / milliliter, of at least 375 micrograms / milliliter, or at least 400 micrograms / milliliter, in the subject.

The doses of the antibodies or fragments thereof of the invention can be repeated, and the administrations can be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days , 2 months, 75 days, 3 months, or at least 6 months.

An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method, the route, and the dose of administration, and the severity of the side effects (see, for example. , Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fia., Dent (2001) Good Laboratory and Good Clinical Practice, Urch P ub I., London, United Kingdom).

The route of administration can be, for example, by topical or cutaneous application, injection or intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intra-arterial, intra-cerebro-spinal, intralesional, or by sustained release or by a implant (see, for example, Sidman et al. (1983) Biopolymers 22: 547-556; Langer et al. (1981) J. Biomed. Mater. Res. 15: 167-277; Langer, (1982) Chem. Tech. 12 : 98-105; Epstein et al. (1985) Proc. Nati. Acad. Sci. USA 82: 3688-3692; Hwang et al. (1980) Proc. Nati. Acad. Sci. USA 77: 4030-4034; the United States of North America Numbers 6,350,466 and 6,316,024). When necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to relieve pain at the site of injection. In addition, pulmonary administration can also be employed, for example, by the use of an inhaler or nebulizer and the formulation with an aerosolizing agent. See, for example, State Patents United States of North America Numbers 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and International Publications of the TCP Numbers WO 92/19244, International Publication Number WO 97/32572, International Publication Number WO 97/44013, and International Publications Nos. WO 98/31346 and WO 99/66903, each of which is incorporated herein by reference. the present as a reference in its entirety.

A composition of the present invention can also be administered by means of one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and / or mode of administration will vary depending on the desired results. The administration routes selected for the antibodies or fragments thereof of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other routes of parenteral administration, for example, by injection or infusion. Parenteral administration can represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intra-peritoneal injection and infusion. , transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal. Alternatively, a composition of the invention can be administered by a non-parenteral route, such as a topical, epidermal or mucosal administration route, for example, intranasal, oral, vaginal, rectal, sublingual or topical. In one embodiment, the antibodies or fragments thereof of the invention are administered by infusion. In another embodiment, the multispecific binding protein to the epitope of the invention is administered subcutaneously.

If the antibodies or fragments thereof of the invention are administered in a controlled release or sustained release system, a pump can be used to achieve controlled or sustained release (see Langer, supra Sefton, (1987) CRC Crit. Ref Biomed, Eng. 14:20, Buchwald et al. (1980) Surgery 88: 507; Saudek et al. (1989) N. Engl. J. Med. 321: 574). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see, for example, Medical Applications of Controlled Release, Langer and Wise (Editors), CRC Pres., Boca Raton, Fia. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (Editors), Wilcy, New York (1984), Ranger and Peppas, (1983), J., Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., (1985), Science 228: 190, During et al., (1989), Ann Neurol. 25: 351, Howard et al., (1989), J. Neurosurg. 7 1: 105); Patents of the United States of North America Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; International Publication of the TCP Number WO 99/15154; and the International Publication of TCP WO 99/20253. Examples of the polymers used in the sustained release formulations include, but are not limited to, poly- (2-hydroxyethyl methacrylate), poly- (methyl methacrylate), poly- (acrylic acid), poly- (ethylene-co-vinyl acetate), poly- (methacrylic acid), poly-glycolides (PLG), poly-anhydrides, poly- (N-vinyl-pyrrolidone), poly- (vinyl alcohol), poly-acrylamide, poly - (ethylene glycol), polylactides (PLA), poly- (lactide-co-glycolides) (PLGA), and poly-ortho-esters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, storage stable, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity to the prophylactic or therapeutic target and, therefore, only a fraction of the systemic dose is required (see, eg, Goodson, in Medical Applications of Controlled Release, supra, volume 2). , pages 1 15-138 (1984)).

Controlled release systems are discussed in the review by Langer ((1990), Science 249: 1527-1533). Any technique known to one skilled in the art can be used to produce sustained release formulations comprising one or more antibodies or fragments thereof of the invention. See, for example, U.S. Patent Number 4,526,938, International Publication of TCP Number WO 91/05548, International Publication of TCP Number WO 96/20698, Ning et al., (1996), Radiotherapy & Oncology 39: 179-189, Song et al. (1995) PDA Journal of Pharmaceutical Science & Technology 50: 372-397, Cleek et al. (1997) Pro. Int'l. Symp. Control. I laughed Bioact. Mater. 24: 853-854, and Lam et al. (1997) Proc. Int'l. Symp. Control Reí. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety.

If the antibodies or fragments thereof of the invention are administered topically, they may be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other well-known form of an expert in the field. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th Edition, Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, typically viscous to semi-solid or solid forms comprising a vehicle or one or more excipients compatible with topical application, and having a dynamic viscosity, in some instances, greater than that of water are employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, balsams, and the like, which, if desired, are sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, pH regulators, or salts) to influence different properties, such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, in some instances, in combination with a solid or liquid inert carrier, it is packaged in a mixture with a pressurized volatile agent (e.g., a gaseous propellant, such as Freon) or in a squeeze bottle. Humidifiers or humectants may also be added to the pharmaceutical compositions and dosage forms if desired. Examples of these additional ingredients are well known in the art.

If the compositions comprising the antibodies or fragments thereof are administered intranasally, they can be formulated in an aerosol, spray, mist, or droplet form. In particular, prophylactic or therapeutic agents to be used in accordance with the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packets or from a nebulizer, with the use of a suitable propellant (for example, dichloro-difluoro-methane, trichloro-fluoro-methane, dichloro-tetrafluoro-ethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to supply a measured quantity. Capsules and cartridges (composed, for example, of gelatin) can be formulated for use in an inhaler or insufflator, containing a powder mixture of the compound and a suitable powder base, such as lactose or starch.

Methods for co-administration or treatment with a The second therapeutic agent, for example, a cytochem, steroid, chemotherapeutic agent, antibiotic, or radiation, are known in the art (see, for example, Hardman et al. (Editors) (2001) Goodman and Gilman, The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill, New York, NY, Poole and Peterson (Editors) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams and Wilkins, Row, Pa .; Chabner and Longo (Editors) (2001) ) Cancer Chemotherapy and Biotherapy, Lippincott, Williams and Wilkins, Row., Pa.). An effective amount of the therapeutic product can decrease the symptoms by at least 10 percent; by at least 20 percent; by at least approximately 30 percent; by at least 40 percent, or by at least 50 percent.

Additional therapies (eg, prophylactic or therapeutic agents) can be administered, which can be administered in combination with the antibodies or fragments thereof of the invention, with less than 5 minutes of separation, with less than 30 minutes of separation, with 1 hour of separation, with about 1 hour of separation, with about 1 to about 2 hours of separation, with about 2 hours to about 3 hours of separation, with about 3 hours to about 4 hours of separation, with about 4 hours of separation. about 5 hours apart, with about 5 hours to about 6 hours apart, with about 6 hours a about 7 hours apart, with about 7 hours to about 8 hours apart, with about 8 hours to about 9 hours apart, with about 9 hours to about 10 hours apart, with about 10 hours to about 11 hours apart, with approximately 1 1 hours to approximately 12 hours of separation, with approximately 12 hours to 18 hours of separation, with 18 hours to 24 hours of separation, with 24 hours to 36 hours of separation, with 36 hours to 48 hours of separation, with 48 hours at 52 hours of separation, with 52 hours at 60 hours of separation, with 60 hours at 72 hours of separation, with 72 hours at 84 hours of separation, with 84 hours at 96 hours of separation, or with 96 hours at 120 hours of separation of the antibodies or fragments thereof of the invention. The two or more therapies can be administered within the same appointment of the patient.

The antibodies or fragments thereof of the invention and the other therapies can be administered cyclically. Cyclic therapy involves the administration of a first therapy (eg, a first prophylactic or therapeutic agent) over a period of time, followed by the administration of a second therapy (eg, a second prophylactic or therapeutic agent) over a period of time. time, optionally, followed by the administration of a third therapy (eg, a third prophylactic or therapeutic agent) over a period of time, and thus successively, and repeating this administration in sequence, that is, the cycle, in order to reduce the development of resistance to one of the therapies, in order to avoid or reduce the side effects of one of the therapies, and / or to improve the effectiveness of therapies.

In certain embodiments, the antibodies or fragments thereof of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. In order to ensure that the therapeutic compounds of the invention cross the blood-brain barrier (BBB) (if desired), they can be formulated, for example, in liposomes. For methods for the preparation of liposomes, see, for example, Patents of the United States of North America Numbers 4,522.81 1; 5,374,548; and 5,399,331. The liposomes may comprise one or more fractions that are selectively transported into specific cells or organs and, consequently, improve the targeted delivery of the drug (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29: 685 ). Exemplary address fractions include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (Bloeman et al. (1995) FEBS Lett. 357: 140; Owais et al. (1995) Antimicrob. Agents Chemother. 180); protein A surfactant receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); page 120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4: 273.

The invention provides protocols for the administration of the pharmaceutical composition comprising the antibodies or fragments thereof of the invention, alone or in combination with other therapies, to a subject in need thereof. Therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention may be administered concomitantly or in sequence to a subject. The therapy (eg, prophylactic or therapeutic agents) of the combination therapies of the present invention can also be administered cyclically. Cyclic therapy involves the administration of a first therapy (eg, a first prophylactic or therapeutic agent) over a period of time, followed by the administration of a second therapy (eg, a second prophylactic or therapeutic agent) over a period of time. time, and repeating this administration in sequence, that is, the cycle, in order to reduce the development of resistance to one of the therapies (for example, agents) in order to avoid or reduce the side effects of one of the therapies (for example, agents), and / or to improve, the effectiveness of therapies.

Therapies (for example, prophylactic agents or Therapeutics) of the combination therapies of the invention can be administered to a subject in a concurrent manner. The term "in a concurrent manner" is not limited to the administration of therapies (eg, prophylactic or therapeutic agents) at exactly the same time, but instead, this means that the pharmaceutical composition, which comprises the antibodies or fragments thereof of the invention, is administered to a subject in a sequence and within a time interval, such that the antibodies of the invention can act together with the other therapies, to provide a greater benefit than if they were administered in another way. For example, each therapy can be administered to a subject at the same time or in sequence in any order at different points of time.; however, if they are not administered at the same time, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate way, and by any suitable route. In different embodiments, therapies (eg, prophylactic or therapeutic agents) are administered to a subject with less than 15 minutes, less than 30 minutes, less than 1 hour apart, approximately 1 hour apart, with approximately 1 hour to about 2 hours apart, with about 2 hours to about 3 hours apart, with about 3 hours to about 4 hours apart, with about 4 hours to about 5 hours apart, with about 5 hours to about 6 hours apart, with about 6 hours to about 7 hours apart, with about 7 hours to about 8 hours apart, with about 8 hours to about 9 hours apart, with about 9 hours to about 10 hours of separation, with about 10 hours to about 11 hours of separation, with about 1 1 hours to about 12 hours of separation, with 24 hours of separation, with 48 hours of separation, with 72 hours of separation of separation, or with 1 week of separation. In other embodiments, the two or more therapies (e.g., prophylactic or therapeutic agents) are administered within the same patient appointment.

The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies may be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents can be administered to a subject by the same or different routes of administration.

The invention having been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not intended to be additionally limitations.

EXAMPLES Example 1: Antibody Methods, Materials and Tracing (i) Cell lines The SK-Br-3, BT-474 and MCF-7 cell lines were purchased from ATCC and routinely maintained in a growth medium supplemented with 10 percent fetal bovine serum (FBS). (ii) Generation of Recombinant Human, Cinomolgus, Mouse, and Rat HER3 Vectors The extracellular domain of murine HER3 was amplified by polymerase chain reaction (PCR) from the mouse brain cDNA (Clontech), and the sequence was verified by a comparison with Refseq NM_010153. Rat HER3 ECD was reverse transcribed from the mRNA of Rata-2 cells, and the sequence was verified by a comparison with NM_017218. The cDNA template of cinomolgus HER3 was generated using RNA from different tissues of cynomolgus (Zyagen Laboratories), and the product of the reverse transcription polymerase chain reaction (RT-PCR) was cloned into pCR®-TOPO -XL (Invitrogen) before the sequencing of both chains. Human HER3 was derived from a human fetal brain cDNA library (Source), and the sequence was verified by a comparison with NM_001982.

To generate labeled recombinant proteins, HER3 human, mouse, rat, and cynomolgus were amplified by polymerase chain reaction (PCR) using Pwo Taq polymerase (Roche Diagnostics). The polymerase chain reaction (PCR) amplified products were gel purified and cloned into a Gateway entry vector pDonR201 (Invitrogen) that had been previously modified to include an N-terminal CD33 leader sequence within the frame and a TAG C-terminal, for example, FLAG TAG. The TAG allows the purification of the monomeric proteins by means of an anti-TAG monoclonal antibody. The target genes were flanked with AttB1 and AttB2, allowing recombination in the registered target vectors adapted by Gateway (eg, pcDNA3.1), using the cloning technology Gateway® (Invitrogen). The recombination reactions were carried out using a Gateway LR reaction with registered target vectors containing a CMV promoter, to create the TAG expression vectors, although any commercially available vector can be used.

Other recombinant HER3 proteins were generated that were fused with the HER3 ECD upstream of a dissociation site of the C-terminal X Factor and the human IgG joint and the Fe domain, to create a Fe-labeled protein. , the different ECDs of HER3 were amplified by polymerase chain reaction (PCR), and cloned into a modified vector (for example, pcDNA3.1) to contain a fusion C-terminal within the framework of the X-Articulation-hFc Factor site.

The generated open reading frame was flanked with the AttB1 and AttB2 sites for further cloning with Gateway® recombinant cloning technology (Invitrogen). An LR Gateway reaction was used to transfer the HER3-Fc to a target expression construct containing a CMV promoter. HER3 point mutation expression constructs were generated using conventional site-directed mutagenesis protocols, and the sequences of the resulting vectors were verified.

Table 2 Generation of HER3 expression vectors. The amino acid numbering of HER3 is based on NP_001973 (human), NP_034283 (mouse), and NP_058914 (rat). (iii) Recombinant HER3 Protein Expression The desired HER3 recombinant proteins were expressed in cell lines derived from HEK293 previously adapted to suspension culture, and grown in a serum-free medium registered from Novartis. Verification of small scale expression was undertaken in transient plate transfection assays of 6 wells based on lipofection. Large-scale production of protein by transient transfection was carried out on a scale of 10 to 20 liters in the WaveMR bioreactor system (Wave Biotech). DNA polyethyleneimine (Polisciences) was used as a plasmid carrier in a ratio of 1: 3 (weight: weight). The cell culture supernatants were harvested at 7-10 days after transfection, and were concentrated by cross-flow filtration and diafiltration before purification. (iv) Purification of Marked Protein The recombinant labeled HER3 proteins (e.g., TAG-HER3) were purified by collecting the cell culture supernatant and concentrating 10 times by cross-flow filtration with a 10 kDa cut-off filter (Fresenius). An anti-TAG column was prepared by coupling an anti-TAG monoclonal antibody to Sepharose 4B activated by CNBr in a final ratio of 10 milligrams of antibody per milliliter of resin. The concentrated supernatant was applied to a 35 milliliter column of anti-Mark at a flow rate of 1 to 2 milliliters / minute. After washing the baseline with PBS, the bound material was eluted with 100 mM glycine (pH 2.7), neutralized, and filtered to sterilize. Protein concentrations were determined by measuring the absorbance at 280 nanometers and converted using a theoretical conversion factor of 0.66 AU / mg. The purified protein was finally characterized by SDS-PAGE, N-terminal sequencing, and LC-MS. (v) Purification of Faith Brand The concentrated cell culture supernatant was applied to a Sepharose Rapid Flow column of 50 milliliters of Protein A, at a flow rate of 1 milliliter / minute. After washing the baseline with PBS, the column was washed with 10 column volumes of 10 mM NaH2P04 / 30 percent (volume / volume) of isopropanol, pH 7.3, followed by 5 column volumes of PBS. Finally, the bound material was eluted with 50 mM Citrate / 140 mM NaCl (pH 2.7), neutralized, and sterilized by filtration. (vi) HuCAL PLATINUM® Paneos For the selection of antibodies that recognize human HER3, multiple panning strategies were employed. Therapeutic antibodies against the human HER3 protein were generated by selecting clones that had high binding affinities, using as the source of the antibody variant proteins, a commercially available phage display library, the MorphoSys HuCAL Platinum® library. The phagemid library is based on the HuCAL® concept (Knappik et al. (2000), J Mol Biol 296: 57-86), and uses the CysDisplay® technology for the display of Fab fragments on the phage surface (International Publication Number W001 / 05950 to Lohning).

For the isolation of anti-HER3 antibodies, they were out of standard panning strategies as well as RapMAT, using approaches of whole cell panning in solid phase, in solution, of whole cells, and differential. (vii) Solid phase panning To identify anti-HER3 antibodies, a variety of solid-phase panning strategies were carried out using different recombinant HER3 proteins. To carry out each round of solid-phase panning, the Maxisorp plates (Nunc) were coated with HER3 protein. The labeled proteins were captured using plates previously coated with anti-Fc antibody (or goat or mouse anti-human IgG, Jackson Immuno Research), anti-Brand antibody, or by passive adsorption. The coated plates were washed with PBS and blocked. The coated plates were washed twice with PBS before the addition of HuCAL Platinum® phage-antibodies for 2 hours at room temperature on a shaker. The bound phages were eluted and added to TG-1 from E. coli, and incubated for phage infection. Subsequently, the infected bacteria were isolated and cultured on agar plates. The colonies were scraped off the plates and the phages were rescued and amplified. Each panning strategy of HER3 comprised individual panning rounds and contained unique antigens, antigen concentrations, and wash restriction. (viii) Panning in the solution phase Each round of panning in the solution phase was carried out using various biotinylated recombinant HER3 proteins in the presence or absence of 1-b 1 neurregulin (R &D Systems). The proteins were biotinylated using the EZ-link sulfo-NHS-LC biotinylation kit (Pierce) according to the manufacturer's instructions. 800 microliters of magnetic beads linked to streptavidin (Dynabeads, Dynal) were washed once with PBS and blocked overnight with Chemiblocker (Chemicon). The HuCAL Platinum® phage-antibodies and the appropriate biotinylated HER3 were incubated in a reaction tube. Streptavidin magnetic beads were added for 20 minutes and harvested with a magnetic particle separator (Dynal). Bound phages were eluted from the Dynabeads by the addition of a regulator containing DTT to each tube, and added to E. coli TG-1. The phage infection was carried out in a manner identical to that described in the solid-phase panning. Each panning strategy of HER3 was comprised of individual panning rounds and contained unique antigens, antigen concentrations, and wash restriction. In order to isolate the antibodies directed towards a specific epitope, competition panes were carried out. In these panning strategies, HER3 was incubated and pre-blocked with a reference antibody prior to the addition of HuCAL Platino® phage-antibodies. As an alternative strategy, reference antibodies were used to specifically elute the phage-antibodies that complexed with HER3. (ix) Cell-based panning For cell panning, the HuCAL Platinum® phage-antibodies were incubated with approximately 107 cells on a rotary apparatus for 2 hours at room temperature, followed by centrifugation. The cell agglomerate was isolated, and the phages were eluted from the cells. The supernatant was collected and added to the E. coli TG-1 culture, continuing with the process described above. Two cell-based strategies were used to identify anti-HER3 antibodies: a) Whole cell panning: In this strategy a variety of intact cell lines were used as the antigens. b) Differential panning of whole cells: In this strategy, the antigens consisted sequentially in cells and recombinant HER3 proteins. The cell-based panning was carried out as described above, while the solid-phase panning protocols were employed when the recombinant proteins were used as antigens. Washes were performed using PBS (2-3X) and PBST (2-3X). (x) Generation of RapMATMR library and paneos In order to increase the binding affinity of the antibody while maintaining the diversity of the library, the production of the second round of pans was introduced both in the solution phase and in the solid phase, in the RapMATMR process, while introducing the production of the third round of whole cell panning and whole cell differential strategies (Prassler et al. (2009) Immunotherapy; 1: 571-583). The RapMATMR libraries were generated by subcloning the inserts encoding Fab from the selected phage through panning in the display vector pMORPH® 25_bla_LHC, and additionally digesting to generate either the RapMATMR H-CDR2 libraries or the L-libraries. CDR3 RapMATMR using specific restriction enzymes The inserts were replaced with the TRIM maturing cassettes (Virnekas et al. (1994) Nucleic Acids Research 22: 5600-5607) for H-CDR2 or L-CDR3 according to the composition of the pool. The library sizes were estimated to be in the range of 8x106-1x108 clones. RapMAT phage antibodies were produced, and were subjected to two additional rounds of solution, solid-phase, or cell-based panning, using the experimental methods. described above.

This extensive panning strategy, which involves an iterative refinement of the library design, was developed specifically to push the scan away from competitive antibodies with the pure ligand, by including the ligand blocking antibodies directly in the panning. Secondly, the process of converting FAB to IgG was adapted to maximize the recovery of the candidate clones, and to ensure that all the selective linkers were profiled in the functional assays. From 44 initial pans, which provided more than x clones, only three families of antibodies exhibited the desired property of blocking signal transduction both dependently and independently of the ligand. Family A that is linked to domains 1 -2 and 2 of isolated Her3. Family B that binds to domains 3-4 isolated, but not to 4 alone; and family C, which is linked to domain 3.

Example 2: Transient expression of anti-HER3 IgGs The HEK293-6E cells adapted in suspension were cultured in a BioWave20. The cells were transiently transfected with the relevant sterile DNA mixture: PEI and further cultured. After transfection, the cells were removed by cross-flow filtration using Fresenius filters. The cell-free material was concentrated by cross-flow filtration using a cut-off filter (Fresenius), and the concentrate was sterilized by filtration through a Stericup filter. The sterile supernatant was stored at 4 ° C.

Example 3: Purification of anti-HER3 IgG The purification of IgG was done in an AKTA 100 Explorer Air chromatography system in a cooling cabinet, using an XK16 / 20 column with 25 milliliters of self-packaging MabSelect SuRe resin (all from GE Healthcare). All flow rates were 3.5 milliliters / minute, except for the load, at a pressure limit of 5 bar. The column was equilibrated with 3 column volumes of phosphate buffered saline (PBS) before loading the filtered fermentation supernatant. The column was washed with phosphate buffered saline (PBS). The IgG was eluted with a pH gradient, starting with citrate / NaCl (pH 4.5), going linearly down to citrate / NaCl (pH 2.5), followed by a constant step of the same pH regulator of 2.5. Fractions containing I g G were pooled and immediately neutralized and sterilized by filtration (Millipore Steriflip, 0.22 microns). The D02eo was measured and the protein concentration was calculated based on the sequence data. The pooled fractions were tested separately to determine agglomeration (SEC-MALS) and purity (SDS-PAGE and MS).

Example 4: Expression and purification of HuCAL®-Fab antibodies in E. coli Expression of the Fab fragments encoded by pMORPH®X9_Fab_MH in TG-1 cells was done in shake flask cultures using the YT medium supplemented with chloramphenicol. The cultures were shaken until the D06oonm reached 0.5. The expression was induced by adding IPTG (isopropyl-3-D-thiogalactopyranoside). The cells were broken using lysozyme. Fab fragments with His6 tag were isolated by means of IMAC (Bio-Rad). The regulator was exchanged with Dulbecco's phosphate buffered (PBS) 1 x (pH 7.2) using PD10 columns. The samples were sterilized by filtration. Protein concentrations were determined by UV spectrophotometry. The purity of the samples was analyzed in 15% SDS-reducing denaturing PAGE. The homogeneity of the Fab preparations was determined in the native state by size exclusion chromatography (HP-SEC) with calibration standards.

Example 5: Measurements of the affinity (KD) of the HER3 antibody by titration of the equilibrium solution (SET) Determination of the affinity in solution was essentially as described above (Friguet et al. (1985), J Immunol Methods 77: 305-19). To improve the sensitivity and accuracy of the SET method, it was transferred from the classical ELISA to the ECL-based technology (Haenel et al. (2005), Anal Biochem 339: 182-84).

To determine the affinity by SET, HER3-Unmarked label (from human, rat, mouse or cynomolgus monkey) was used.

The data was evaluated with Xlfit software (ID Business Solutions) applying particularly adapted adjustment models. For the determination of the KD of each IgG the following model was used (modified according to Piehler et al. (Piehler et al. (1997) J Immunol Methods 201: 189-206).

[IgG]: total concentration of IgG applied. x: total concentration of applied soluble antigen (binding sites).

Bmax: maximum IgG signal without antigen.

KD: affinity.

Example 6: Determination of cell-antibody binding by means of FACS The binding of the antibodies to the endogenous human antigen expressed on human cancer cells was evaluated by FACS. To determine the EC50 values of the antibody, SK-Br-3 cells were harvested with acutase and diluted to 1 x 106 cells / milliliter in FACS regulator (PBS / 3% FBS / 0.2% NaN3). One x 105 cells / well was added to each well of a 96-well plate (Nunc), and centrifuged at 210 g for 5 minutes at 4 ° C before removing the supernatant. Serial dilutions of the test antibodies (diluted in steps of 1: 4 dilution with FACS regulator) were added to the agglomerated cells, and incubated for 1 hour on ice. The cells were washed and agglomerated three times with 100 microliters of FACS regulator. Anti-human goat IgG conjugated to PE (Jackson ImmunoResearch), diluted at 1/200 with FACS regulator, was added to the cells and incubated on ice for 1 hour. Additional washing steps were made three times with 100 microliters of FACS regulator, followed by centrifugation steps at 210 g for 5 minutes at 4 ° C. Finally, the cells were resuspended in 200 microliters of FACS regulator and the fluorescence values were measured with FACSArray (BD Biosciences). The amount of anti-HER3 antibody bound at the cell surface was determined by measuring the fluorescence of the average channel.

Example 7: Domain linkage and HER3 mutant The 96-well Maxlsorp plates (Nunc) were coated overnight with 200 nanograms of the appropriate recombinant human protein (HER3-Mark, D1-2-Mark, D2-Mark, D3-4-Mark, D4-Mark, HER3 K267A -Mark, HER3 L268A-Mark, HER3 K267A / L268A and a marked irrelevant control). All wells were then washed with phosphate buffered saline (PBS) / 0.1 percent Tween-20, were blocked with phosphate buffered serum (PBS) / 1 percent bovine serum albumin (BSA) / 0.1 percent Tween-20, and washed with phosphate buffered saline (PBS) / 0.1 Tween-20 hundred. Anti-HER3 antibodies were added to the relevant wells to a final concentration of 10 micrograms / milliliter, and incubated at room temperature. Plates were washed with 0.1% phosphate-buffered serum (PBS) / 0.1% Tween-20 prior to the addition of the appropriate peroxidase-linked detection antibody diluted 1 / 10,000 in phosphate-buffered serum (PBS) / bovine serum albumin. (BSA) at 1 percent / Tween-20 at 0.1 percent. The detection antibodies used were goat anti-mouse (Pierce, 31432), rabbit anti-goat (Pierce, 31402), and goat anti-human (Pierce, 31412). Plates were incubated at room temperature before washing with 0.1 percent phosphate buffered saline (PBS) / Tween-20. 100 microliters of the TMB substrate solution (3,3 ', 5,5'-tetramethyl-benzidine) (BioFx) was added to all wells before stopping the reaction with 50 microliters of H2SO4 at 2.5 percent. The degree of binding of the HER3 antibody to each recombinant protein was determined by measurement of OD 45o using a SpectraMax plate reader (Molecular Devices). Where appropriate, dose response curves were analyzed using Graphpad Prism.

Example 8: Antibody cross-competition by ELISA Antibody A was coated in a constant amount on Maxisorp plates, and was tested to determine the competition of HER3 binding with increasing amounts of antibody B in solution. Maxisorp plates were coated with 24 nanograms / well of antibody A in phosphate buffered serum (PBS), incubated overnight at 4 ° C, and then washed with PBST. The plates were blocked with 3 percent BSA / PBS for 1 hour at room temperature. Antibody B was titrated in 1: 3 steps and incubated in a molar excess with HER3-biotinylated label for 1 hour at room temperature in solution. The HER3 / antibody B complexes were then added to the plate coated with antibody A for 30 minutes, and the bound complexes were detected by quantifying the amount of HER3-biotinylated label. The blocked plates were subsequently washed with PBST, the previously formed HER3 / antibody B complexes were added, and incubated for 30 minutes at room temperature with gentle agitation. The plates were subsequently washed with an excess of PBST, and incubated for 1 hour with streptavidin-AP diluted 1: 5000 in 1 percent bovine serum albumin (BSA) / 0.05 percent Tween 20 / phosphate buffered serum (PBS). The plates were washed with PBST, a solution of AttoPhos (1: 5 diluted in H2O) was added, and the fluorescent signals were measured at 535 nanometers followed by excitation at 430 nanometers.

If antibody A did not compete with antibody B to bind to HER3, then a high level of HER3 was detected. In contrast, for competitive antibodies or antibodies with partially overlapping epitopes, HER3 signals decreased significantly when compared to IgG controls.

Example 9: In vitro cell tests of Fosfo-HER3.

The MCF-7 cells were routinely maintained in DMEM / F12, 15 mM HEPES, L-glutamine, fetal bovine serum (FBS) at 10 percent, BT474 in DMEM, 10 percent fetal bovine serum and SK-Br-3 in the 5th of McCoy, fetal bovine serum (FBS) at 10 percent, L-glutamine 1.5 mM. The sub-confluent cells were trypsinized, washed with phosphate-buffered serum (PBS), and diluted to 5 x 10 5 cells / milliliter. Then 100 microliters of cell suspension was added to each well of a 96 well flat bottom plate (Nunc), to give a final density of 5 x 10 4 cells / well. The MCF7 cells were allowed to adhere for approximately 3 hours before exchanging the medium for a starvation medium containing 0.5 percent fetal bovine serum (FBS). Afterwards, all the plates were incubated overnight at 37 ° C before treatment with the appropriate concentration of HER3 antibodies for 80 minutes at 37 ° C. The MCF7 cells were treated with 50 nanograms / milliliter of NRG 1 during the final 20 minutes to stimulate the phosphorylation of HER3 and AKT while the BT474 / SK-Br-3 cells did not require any additional stimulation. All media were gently aspirated and the cells were washed with ice-cold phosphate buffered (PBS) containing 1 mM CaCl 2 and 0.5 mM MgCl 2 (Gibco). Cells were used by adding 50 microliters of ice cold lysis buffer (20 mM Tris (pH 8.0) / 137 mM NaCl / 10 percent glycerol / 2mM EDTA / 1 percent NP-40 / sodium orthovanadate 1 mM / 1x Fosfo-Stop / mini-protease inhibitors Complete 1 x (Roche) / 0.1 mM PMSF), and incubated on ice with shaking for 30 minutes. Then, the Used ones were collected and centrifuged at 1800 g for 15 minutes at 4 ° C to remove cellular debris.

HER3 capture plates were generated using a carbon plate (Mesoscale Discovery) coated overnight at 4 ° C with 20 microliters of capture antibody MAB3481 (R & D Systems) diluted to 4 micrograms / milliliter in phosphate buffered serum ( PBS), and were subsequently blocked with 3 percent bovine serum albumin in Tris 1 x (Mesoscale Discovery) / 0.1 percent Tween-20 buffer. The HER3 was captured by adding the appropriate amount of the Used, and incubating the plate at room temperature for one hour with shaking before aspirating the Used one, and the wells were washed with regulator of Tris 1 x (Mesoscale Discovery) / Tween-20 to 0.1 percent. The phosphorylated HER3 was detected using 1: 8000 anti-pY1 197 antibody (Cell Signaling) prepared in 3 percent milk / 1 x Tris / 0.1 percent Tween-20, incubating with shaking at room temperature for 1 hour. The wells were washed four times with 1 x Tris / 0.1% Tween-20, and the phosphorylated proteins were detected by incubation with the goat anti-rabbit antibody labeled with S-Mark (# R32AB) diluted in the regulator. of blocking at 3 percent for one hour at room temperature. Each well was aspirated and washed four times with 1 x Tris / 0.1 percent Tween-20, before adding 20 microliters of the T-reading regulator with surfactant (Mesoscale Discovery), and the signal was quantified using a Mesoscale Sector Imager.

Example 10: In vitro cell assays of phospho-Akt (S473).

Subconfluent MCF7, SK-Br-3 and BT-474 cells were grown in the complete medium, harvested with acutase (PAA Laboratories), and resuspended in the appropriate growth medium at a final concentration of 5 x 105 cells / milliliter. Then 100 microliters of cell suspension was added to each well of a 96 well flat bottom plate (Nunc), to produce a final density of 5 x 10 4 cells / well. The MCF7 cells were allowed to adhere for approximately 3 hours before exchanging the medium for a starvation medium containing 0.5 percent fetal bovine serum (FBS). All plates were incubated overnight at 37 ° C before treatment with the appropriate concentration of HER3 antibodies for 80 minutes at 37 ° C. The MCF7 cells were treated with 50 nanograms / milliliter of NRG1 for the final 20 minutes to stimulate the phosphorylation of HER3 and AKT while the SK-Br-3 cells did not require additional stimulation. All medium was gently aspirated and the cells were washed with ice-cold phosphate buffered serum (PBS) containing 1 mM CaCl 2 and 0.5 mM MgCl 2 (Gibco). Cells were used by adding 50 microliters of ice-cold lysis buffer (20 mM Tris (pH 8.0) / 137 mM NaCl / 10 percent glycerol / 2 mM EDTA / 1 percent NP-40 / sodium orthovanadate 1 mM / aprotinin (10 micrograms / milliliter) / leupeptin (10 micrograms / milliliter)), and incubated on ice with shaking for 30 minutes. Then, the Used ones were collected and centrifuged at 1800 g for 15 minutes at 4 ° C to remove all cell debris. 20 microliters of the Used were added to a 384-well Phospho-Akt Multi-Spot carbon plate (Mesoscale Discovery), which had previously been blocked with 3 percent bovine serum albumin (BSA) / 1x Tris / 20 Tween-20 albumen. 0.1 percent. The plate was incubated at room temperature for two hours with shaking before aspirating the Used, and the wells were washed four times with Tris 1 x (Mesoscale Discovery) / 0.1% Tween-20 buffer. Phosphorylated Akt was detected using 20 microliters of anti-phospho-Akt (S473) SULFO-TAG antibody (Mesoscale Discovery) diluted 50 fold in bovine serum albumin (BSA) at 1 percent / Tris 1 x / 0.1 percent Tween-20, incubating with stirring at room temperature for 2 hours. The wells were washed four times with 1 x Tris / 0.1 percent Tween-20, before adding 20 microliters of the T-reading regulator with surfactant (Mesoscale Discovery), and the signal was quantified using a Mesoscale Sector Imager.

Example 11: Proliferation tests of the cell line.

SK-Br-3 cells were routinely cultured in a modified McCoy 5A medium supplemented with 10 percent fetal bovine serum, and BT-474 cells were cultured in DMEM supplemented with 10 percent fetal bovine serum (FBS). . Subconfluent cells were trypsinized, washed with phosphate buffered saline (PBS), diluted to 5 x 10 4 cells / milliliter with the growth medium, and seeded in black 96-well clear bottom plates (Costar 3904) to a density of 5,000 cells / well. Cells were incubated overnight at 37 ° C before adding the appropriate concentration of HER3 antibody (typical final concentrations of 10 or 1 microgram / milliliter). The plates were returned to the incubator for 6 days before determining cell viability using the CelITiter-Glo (Promega). To each well, 100 microliters of the CelITiter-Glo solution was added, and incubated at room temperature with gentle agitation for 10 minutes. The amount of luminescence was determined using a SpectraMax plate reader (Molecular Devices). The magnitude of growth inhibition obtained with each antibody was calculated by comparing the luminescence values obtained with each HER3 antibody with a standard isotype control antibody.

For proliferation tests, MCF-7 cells were routinely cultured in DM EM / F12 (1: 1) containing 4 mM L-glutamine / 15 mM HEPES / 10 percent FBS. Subconfluent cells were trypsinized, washed with phosphate buffered saline (PBS) and diluted to 1x105 cells / milliliter with DMEM / F12 (1: 1), containing 4mM L-glutamine / 15mM HEPES / 10 micro-grams / milliliter of human transferrin / bovine serum albumin (BSA) at 0.2 percent. Cells were seeded in black 96-well clear bottom plates (Costar) at a density of 5,000 cells / well. Then the appropriate concentration of the HER3 antibody was added (typical final concentrations of 10 or 1 microgram / milliliter). Also 10 nanograms / milliliter of the EGF domain of NRGI -bI (R &D Systems) were added to the appropriate wells to stimulate cell growth. The plates were returned to the incubator for 6 days before determining cell viability using CelITiter-Glo (Promega). The magnitude of the growth inhibition obtained with each antibody was calculated by subtracting the background luminescence values (without neurregulin) and comparing the resulting values obtained with each anti-HER3 antibody with a standard control antibody of the isotype.

Example 12: BxPC3 efficacy studies BxPC3 cells were cultured in an RPMI-1640 medium that contained 10 percent fetal bovine serum inactivated by heat without antibiotics until the time of implantation.

Subcutaneously nude nu / nu female Balb / C mice (Harán Laboratories), 10 x 10 6 cells were implanted in a mixture of 50 percent phosphate buffered saline with 50 percent Matrigel. The total injection volume containing cells in suspension was 200 liters. Once the tumors reached a size of approximately 200 mm3, the animals were enrolled in the efficacy study. In general, a total of 10 animals were enrolled per group in the studies. Animals were excluded from enrollment if they exhibited unusual tumor growth characteristics prior to enrollment.

The animals were dosed intravenously by injection into the lateral vein of the tail. The animals were in a program of 20 milligrams / kilogram, twice a week, for the duration of the study. Tumor volume and T / C values were calculated as detailed for the BT-474 studies.

Example 13: Efficiency studies of BT-474 in vivo BT-474 cells were cultured in a DMEM medium containing 10 percent fetal bovine serum inactivated by heat without antibiotics until the time of implantation.

One day before the inoculation of the cells, atomic nu / nu Balb / C mice (Harán Laboratories) were implanted subcutaneously with an agglomerate of 17 -estradiol of sustained release (Innovative Research of America) to maintain the estrogen levels in serum. One day after implantation of the 173-estradiol pellet, 5 x 10 6 cells were orthotopically injected into the 4th mammary fat pad in a suspension containing 50% phenol-free Matrigel (BD Biosciences) in a saline solution. balanced by Hank. The total injection volume containing the cells in suspension was 200 liters. 20 days after the implantation of the cells, animals with a tumor volume of approximately 200 mm3 were enrolled in the efficacy study. In general, a total of 10 animals were enrolled per group in the efficacy studies.

For studies with the individual agent, the animals were dosed intravenously by injection into the lateral vein of the tail with the control IgG or with MOR13759. The animals were with a dosing program of 20 milli grams / kilogram, twice a week, for the duration of the study. For the duration of the studies, the tumor volume was measured by calibration twice a week. The values of the treatment / control percentage (T / C) were calculated using the following formula: % of T / C = 100 x DT / DO if DT > 0 where: T = mean tumor volume of the group treated with the drug on the final day of the study; DT = mean tumor volume of the group treated with the drug on the final day of the study - mean tumor volume of the group treated with the drug on the initial day of dosing; C = mean tumor volume of the control group on the final day of the study; Y AC = mean tumor volume of the control group on the final day of the study - mean tumor volume of the group treated with the drug on the initial day of dosing.

Body weight was measured twice a week and the dose was adjusted to body weight. The percentage of change in body weight was calculated as (BWactuai - BW¡nic¡ai) / (B Winic¡ai) x 100. The data are presented as the percentage of change in body weight from the start day of the treatment.

All data were expressed with the average + standard error of the average (SEM). The delta volume of the tumor and the body weight were used for the statistical analysis. Comparisons were made between the groups using a unidirectional ANOVA followed by a Tukey post hoc. For all statistical evaluations, the level of significance was adjusted to p < 0.05. The significance compared with the vehicle control group is reported. Results and Discussion Collectively, these results show that a class of antibodies bind to the amino acid residues within domain 2. The binding of these antibodies inhibits signaling both ligand-dependent and ligand-independent. (i) Affinity determination The affinity of the antibody was determined by titration in equilibrium in solution (SET) as described above. The results are presented in summary form in Table 3, and the example titration curves for MOR12616 and MOR12925 are contained in Figure 1. The data indicate that a number of antibodies that bound strongly to human HER3, cynomolgus, were identified. , of rat, and of murine.

Table 3 KD values of anti-HER3 IgGs as determined by equilibrium titration in solution (SET). Hu (human), Cy (cynomolgus), Mu (murine) and ra (rat) (i i) Determination of EC50 in SK-Br-3 cells The ability of the antibodies identified to bind to cells expressing HER3 was determined by calculating the EC50 values for their binding to the cell line amplified with HER2, SK-Br-3 (see Figure 2 and Table 4).

Table 4 FACS ECS0 values of anti-HER3 IgG in the cells (iii) Link to the domain of HER3 A subset of anti-HER3 antibodies was characterized for its ability to bind to the various extracellular domains of human HER3 in an ELISA assay. To achieve this, the extracellular domain of HER3 was divided into its four constitutive domains and several combinations of said domains were cloned, purified and purified as independent proteins as described above. Using this strategy, the following domains are successfully generated as soluble proteins: domains 1 and 2 (D1 -2), domain 2 (D2), domains 3 and 4 (D3-4) and domain 4 (D4). The integrity of each isolated domain was previously confirmed using a panel of internally generated antibodies as the positive controls.

As shown in Figure 3, it was observed that both MOR12616 and MOR12925 were linked to the extracellular domain of HER3, to the isolated D1 -2, and to the isolated D2 protein. No link was observed with the D3-4 or D4 proteins. These binding data suggest that this family of antibodies recognizes an epitope contained primarily within domain 2. To further confirm the epitope, we determine the impact of mutating the residues within D2 after antibody binding. As determined by both the binding ELISA (Figure 4) and the SET (Table 5), the mutation of Lysine268 to alanine severely altered the binding of the antibody, confirming, therefore, that the binding epitope is contained within the domain 2. .

Table 5 K0 values of the anti-HER3 IgG binding to the mutant forms of HER3, as determined by equilibrium titration in solution (SET). v) Epitope competition ELISA In order to further refine the epitope of this class of anti-HER3 antibodies, we carried out epitope competition studies on a subset of antibodies against a number of registered anti-HER3 antibodies whose epitopes had been previously characterized. The epitope competition experiments consist of antibody A (eg, MOR12925 or MOR12616) which is immobilized on a plate, and its ability to capture HER3 / antibody B complexes from a solution. If antibody A does not compete with antibody B to bind to H ER3, then HER3 complexes are captured from a solution. In contrast, if antibody A possesses an identical or overlapping epitope to antibody B, then HER3 complexes can not be captured. Using this method, the alloestheric competitors can also be identified. In this instance, the binding of antibody B to HER3 could induce a conformational change that masks the epitope of antibody A. Therefore, it can appear that antibody A and antibody B compete directly even when their HER3 binding residues can be disconnected from each other.

The example epitope competition data for MOR12925 and MOR 12616 are illustrated in Figure 5. As can be seen from the data, both MOR12925 and MOR12616 effectively cross-compete to bind to HER3, thereby demonstrating that these highly related antibodies probably bind to the same epitope of H ER3. Cross-competition was also observed with an antibody (D2 / 4) whose epitope has been previously mapped into the residues contained within domains 2 and 4. It is interesting that no competition was observed with an antibody (D4) that binds to the domain 4 of HER3 isolated. These data suggest that both MOR12925 and MOR12616 bind to an epitope contained within domain 2, which is consistent with our ELISA Previous domain link. Because antibody D2 / 4 has been shown to interact with amino acid residues 265-277, 315 within domain 2 of HER3, it can be inferred that some of these residues may also be critical for the binding of MOR12925 and MOR12616. (vi) Inhibition of cell signaling In order to ascertain the effect of anti-HER3 antibodies on the ligand-dependent HER3 activity, MCF7 cells were incubated with IgG prior to neurregulin stimulation. The example inhibition curves are illustrated in Figure 6 and are presented in summary form in Table 6. The effect of anti-HER3 antibodies on HER2 activation mediated by HER2 was also studied, using the cell lines amplified by HER2. : SK-Br-3 and BT474 (Figure 7 and Table 6).

Table 6 IC50 of pHER3 and values of degree of inhibition of anti-HER3 IgG in MCF7, BT474 and SK-Br-3 cells To determine if the inhibition of HER3 activity impacted the Akt downstream cell signaling, also Phosphorylation of MCF7 cells stimulated by NRG and SK-Br-3 / BT474 cells amplified by HER2 was measured following treatment with anti-HER3 antibodies (see Figure 6, Figure 7, and Table 7).

TABLE 7 IC50 of pAkt (S473) and values of the degree of inhibition of anti-HER3 IgG in SK-Br-3, BT-474 and MCF7 cells In summary MOR12509, MOR12510, MOR12616, MOR12923, MOR12924, MOR12925, MOR13750, MOR13752, MOR13754, MOR13755, MOR13756, MOR13758, MOR13759, MOR13761, MOR13762, MOR13763, MOR13765, MOR13766, MOR13767, MOR13768, MOR13867, MOR13868, MOR13869, MOR13870, MOR13871, MOR14535 and MOR14536 are each capable of inhibiting the cellular activity of HER3 in a manner both ligand-dependent and ligand-independent. (vii) Inhibition of proliferation Because MOR1 2509, MOR125 0, MOR12616, MOR12923, MOR12924, MOR12925, MOR13750, MOR13752, MOR13754, MOR13755, MOR13756, MOR13758, MOR13759, MOR13761, MOR13762, MOR13763, MOR13765, MOR13766, MOR13767, MOR13768, MOR 13867, MOR13868, MOR13869, MOR13870, MOR 13871, MOR14535 and MOR 14536 inhibited HER3 activity and downstream signaling were tested for their ability to block cell growth independent and ligand-independent in vitro (the example data are shown in Figure 8 and are presented in summary form in Table 8). The anti-HER3 antibodies tested were effective inhibitors of cell proliferation, confirming their ability to inhibit ligand-independent and HER3-driven phenotypes.

Table 8 Inhibition of proliferation following treatment with anti-HER3 IgG in cells SK-Br-3, BT-474 and MCF7 (viii) Inhibition of tumor growth in vivo In order to determine the in vivo activity of the described anti-HER3 antibody, MOR13759 was tested in both tumor models BxPC3 and BT-474. Repeated treatment with MOR13759 gave a regression of 29.1 percent for the BxPC3 model (Figure 9A). Treatment of model BT474 with MOR13759 resulted in 45 percent inhibition of tumor growth (T / C) (Figure 9B).

Incorporation as reference All references cited in the present application, including patents, patent applications, articles, textbooks, and the like, and references cited therein, to the extent that they have not been incorporated, are incorporated by reference into the present application in its entirety.

Equivalents The above written description is considered sufficient to allow a person skilled in the art to practice the invention. The above description and the examples present in detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the description appears in the text, the invention can be practiced in many forms and the invention should be considered in accordance with the appended claims and any equivalents thereof.

Claims (36)

1. CLAIMS 1. An isolated antibody or a fragment thereof that recognizes an epitope of a HER3 receptor, wherein the epitope comprises amino acid residues 208-328 within domain 2 of the HER3 receptor, wherein the antibody or a fragment thereof recognizes at least the amino acid residue 268 within domain 2, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent. 2. The isolated antibody or fragment thereof of claim 1, wherein the epitope is selected from the group consisting of a linear epitope, a non-linear epitope, and a conformational epitope. 3. The isolated antibody or fragment thereof of claim 1, wherein the antibody or a fragment thereof binds to an inactive state of the HER3 receptor. 4. The isolated antibody or fragment thereof of claim 1, wherein the binding of the HER3 ligand to the ligand binding site fails to activate signal transduction of HER3. 5. The isolated antibody or fragment thereof of claim 1, wherein a HER3 ligand can be linked in a concurrent manner to the ligand binding site on the HER3 receptor. 6. The isolated antibody or fragment thereof of claim 5, wherein the HER3 ligand is selected from the group consisting of neurregulin 1 (NRG), neurregulin 2, beta-cellulin, epidermal growth factor that binds to heparin , and epiregulin. 7. The isolated antibody or fragment thereof of claim 1, wherein at least amino acid residue 268 (within domain 2) affects the bond in domain 2, thereby blocking the binding of the antibody or antibody fragment. 8. The isolated antibody or the fragment thereof, wherein the antibody or a fragment thereof has a characteristic selected from the group consisting of destabilizing HER3 in such a manner as to be susceptible to degradation, accelerating the sub-regulation of HER3 of the cell surface, inhibit dimerization with other HER receptors, and generate an unnatural HER3 dimer that is susceptible to proteolytic degradation or that is incapable of dimerizing with other receptor tyrosine kinases. 9. The isolated antibody or fragment thereof of claim 1, wherein the binding of the antibody or a fragment thereof to the HER3 receptor in the absence of a HER3 ligand, reduces the ligand-independent formation of a protein complex of HER2- HER3 in a cell that expresses HER2 and HER3. 10. The isolated antibody or the fragment thereof of the claim 9, wherein the HER3 receptor fails to dimerize with the HER2 receptor to form a protein complex of HER2-HER3. eleven . The isolated antibody or fragment thereof of claim 10, wherein the failure to form a HER2-HER3 protein complex prevents the activation of signal transduction. 12. The isolated antibody or fragment thereof of claim 9, wherein the antibody or a fragment thereof inhibits the phosphorylation of HER3 as assessed by a phosphorylation assay independent of the HER3 ligand. 13. The isolated antibody or fragment thereof of claim 12, wherein the HER3 ligand-independent phosphorylation assay utilizes cells amplified by HER2, wherein the cells amplified by HER2 are the SK-Br-3 and BT-474 cells. 14. The isolated antibody or fragment thereof of claim 1, wherein the binding of the antibody or a fragment thereof to the HER3 receptor in the presence of a HER3 ligand, reduces the formation of a dependent HER2-HER3 protein complex. of the ligand in a cell that expresses HER2 and HER3. 15. The isolated antibody or fragment thereof of claim 12, wherein the HER3 receptor fails to dimerize with the HER2 receptor in the presence of a HER3 ligand to form a protein complex of HER2-HER3. 16. The isolated antibody or fragment thereof of claim 13, wherein failure to form a HER2-HER3 protein complex prevents the activation of signal transduction. 17. The isolated antibody or fragment thereof of claim 14, wherein the antibody or a fragment thereof inhibits the phosphorylation of HER3 as assessed by the HER3 ligand-dependent phosphorylation assay. 18. The isolated antibody or fragment thereof of claim 17, wherein the HER3 ligand-dependent phosphorylation assay utilizes MCF7 cells stimulated in the presence of neurregulin (NRG). 19. The isolated antibody or fragment thereof of claim 1, wherein the antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, and a synthetic antibody. 20. An isolated antibody or a fragment thereof that recognizes an epitope of a HER3 receptor within domain 2 of the HER3 receptor, wherein the epitope comprises amino acid residues 208-328 within domain 2 of the HER3 receptor, wherein the antibody or a fragment thereof recognizes at least amino acid residue 268 within domain 2, and wherein the antibody or a fragment thereof has a dissociation (KD) of at least 1 × 10 7 M 1, 108 M 1, 109 M · 101 ° M 1, 1011 M 1, 1012 M 1, 1013 M 1, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent. 21. The isolated antibody or fragment thereof of claim 20, wherein the antibody or a fragment thereof inhibits the phosphorylation of HER3 as measured by an in vitro phosphorylation assay selected from the group consisting of phospho-HER3 and phospho-phosphorylation. -Akt. 22. The isolated antibody or fragment thereof of claim 20, wherein the antibody or a fragment thereof binds to the same epitope as an antibody described in Table 1 . 23. The isolated antibody or fragment thereof of claim 20, wherein the isolated antibody or fragment thereof cross-competes with an antibody described in Table 1. 24. The isolated antibody or fragment thereof of claim 20, wherein the fragment of an antibody is selected from the group consisting of; Fab, F (ab2) ', F (ab) 2', scFv, VHH, VH, VL, dAbs. 25. A pharmaceutical composition, which comprises an antibody or a fragment thereof and a pharmaceutically acceptable carrier, wherein the antibody or a fragment thereof binds to a HER3 receptor comprising amino acid residues 208-328 within domain 2 of the recipient HER3, in wherein the antibody or a fragment thereof recognizes at least amino acid residue 268 within domain 2, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent. 26. The pharmaceutical composition of claim 25, which further comprises an additional therapeutic agent. 27. The pharmaceutical composition of claim 26, wherein the additional therapeutic agent is selected from the group consisting of a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, an mTOR inhibitor and an inhibitor. of PI3 kinase. 28. The pharmaceutical composition of claim 27, wherein the additional therapeutic agent is an HER1 inhibitor selected from the group consisting of Matuzumab (EMD72000), Erbitux® / Cetuximab, Vectibix® / Panitumumab, mAb 806, Nimotuzumab, Iressa® / Gefitinib, CI-1033 (PD183805), Lapatinib (GW-572016), Tykerb® / Lapatinib Ditosylate, Tarceva® / Erlotinib HCL (OSI-774), PKI-166, and Tovok®; an HER2 inhibitor selected from the group consisting of Pertuzumab, Trastuzumab, MM-11, neratinib, lapatinib or lapatinib ditosylate / Tykerb®; an HER3 inhibitor selected from the group consisting of MM-121, MM-11, IB4C3, 2DI D12 (U3 Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech), MOR10703 ( Novartis), and small molecules that inhibit HER3; and a HER4 inhibitor. 29. The pharmaceutical composition of claim 27, wherein the additional therapeutic agent is an mTOR inhibitor selected from the group consisting of Temsirolimus / Torisel®, ridaforolimus / Deforolimus, AP23573, MK8669, everolimus / Affi nitor®. 30. The pharmaceutical composition of claim 27, wherein the additional therapeutic agent is a PI3 kinase inhibitor selected from the group consisting of GDC 0941, BEZ235, BM K120 and BYL719. 31 A method for the treatment of a cancer, which comprises selecting a subject having a cancer expressing HER3, administering to the subject, an effective amount of a composition comprising an antibody or a fragment thereof as disclosed in the Table 1, wherein the antibody or a fragment thereof recognizes an epitope of a HER3 receptor comprising amino acid residues 208-328 within domain 2 of the HER3 receptor, wherein the antibody or a fragment thereof recognizes at least the residue of amino acid 268 within domain 2, and wherein the antibody or a fragment thereof blocks signal transduction both ligand-dependent and ligand-independent. 32. The method of claim 31, wherein the subject is a human being, and the cancer is selected from the group consisting of breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors, schwannoma, head and neck cancer, cancer of bladder, esophageal cancer, Barrett's esophageal cancer, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis. 33. The method of claim 31, wherein the cancer is breast cancer. 34. An antibody or a fragment thereof of claims 1 to 30, for use in the treatment of a cancer mediated by a pathway of signal transduction or signal transduction independent of the HER3 ligand. 35. An antibody or fragment thereof of claims 1 to 30, for use as a medicament. 36. The use of an antibody or fragment thereof of claims 1 to 30, for the manufacture of a medicament for the treatment of a cancer mediated by a pathway of signal transduction or signal transduction independent of the ligand of HER3, selected from the group consisting of breast cancer, colo-rectal cancer, lung cancer, multiple myeloma, ovarian cancer, cancer liver, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral tumors of the nerve sheath, schwannoma, head and neck cancer, bladder cancer, esophageal cancer, esophageal cancer Barretts, glioblastoma, soft tissue clear cell sarcoma, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia, and endometriosis.